Transformed plant with Bacillus thuringiensis toxin gene

ABSTRACT

A Bacillus thuringiensis strain isolate, designated B.t. strain EG5847, exhibits insecticidal activity against lepidopteran insects. Two novel toxin genes from B.t. strain EG5847 designated cryET4 and cryET5 produce insecticidal proteins with activity against a broad spectrum of insects of the order Lepidoptera, The cryET4 gene has a nucleotide base sequence shown in FIG. 1 and listed in SEQ ID NO:1 and produces a CryET4 gene product having the deduced amino acid sequence shown in FIG. 1 and listed in SEQ ID NO:2. The cryET5 gene has a nucleotide base sequence shown in FIG. 2 and listed in SEQ ID NO:3 and produces a CryET5 gene product having the deduced amino acid sequence shown in FIG. 2 and listed in SEQ ID NO:4.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a division of U.S. patent application Ser. No.08/474,038, filed Jun. 7, 1995, now U.S. Pat. No. 5,679,343, which is adivision of U.S. patent application Ser. No. 08/176,865, filed Dec. 30,1993, now U.S. Pat. No. 5,616,319, which is a division of U.S. patentapplication Ser. No. 08/100,709, filed Jul. 29, 1993, now U.S. Pat. No.5,322,687.

FIELD OF THE INVENTION

The present invention relates to lepidopteran-toxic proteins and thegenes coding therefor. In particular, the present invention is directedto genes designated as cryET4 (SEQ ID NO:1) and cryET5 (SEQ ID NO:3) andtheir proteins designated respectively as CryET4 (SEQ ID NO:2) andCryET5 (SEQ ID NO:4).

BACKGROUND OF THE INVENTION

Bacillus thuringiensis (commonly known as B.t.) is a gram-positive soilbacterium that often produces cellular inclusions during sporulationwhich are specifically toxic to certain orders and species of insects.Many different strains of B.t. have been shown to produce theseinclusions of insecticidal crystal protein (ICP). Compositions includingB.t. strains which produce insecticidal proteins have been commerciallyavailable and used as environmentally acceptable insecticides becausethey are quite toxic to the specific target insect, but are harmless toplants and other non-targeted organisms.

B.t. ICP toxins are active in the insect only after ingestion. Afteringestion by an insect, the alkaline pH and proteolytic enzymes in themid-gut solubilize the crystal allowing the release of the toxiccomponents. These toxic components disrupt the mid-gut cells resultingin cessation of feeding and, eventually, death of the insect. B.t. hasproven to be an effective and environmentally safe insecticide indealing with various insect pests.

A number of genes encoding crystal proteins have been cloned from manystrains of B.t. A good overview is set forth in H. Hofte and H. R.Whiteley, Microbiol. Rev., 53, pp. 242-255 (1989), hereinafter "Hofteand Whiteley (1989)." This reference provides a good overview of thegenes and proteins obtained from B.t. and their uses, adopts anomenclature and classification scheme for B.t. genes and proteins, andhas an extensive bibliography.

The nucleotide sequences of ICP genes responsible for a given crystalphenotype and active against the same insect order are generally morerelated, i.e., more homologous, than are the nucleotide sequences ofB.t. genes encoding delta-endotoxin proteins active against differentorders of insects. Hofte and Whiteley (1989) defines an orderedclassification of genes encoding B.t. delta-endotoxin proteins based onhomology of delta-endotoxin amino acid sequences, as well assimilarities in insecticidal activity; a subranking has also beenestablished based upon further refinement of sequence relationship. Asnoted by Hofte and Whiteley (1989), the majority of insecticidal B.t.strains are active against insects of the order Lepidoptera, i.e.,caterpillar insects. Insecticidal crystal proteins specifically activeagainst Lepidoptera have been designated CryI proteins. These ICPs areencoded by cryI genes. Other B.t. strains produce different classes ofcrystal proteins, e.g., CryII proteins are active against lepidopteranand (for CryIIA) dipteran insects; CryIII proteins are insecticidal toinsects of the order Coleoptera, i.e., beetles; and CryIV proteins areactive against insects of the order Diptera, i.e., flies and mosquitoes.A compilation of the amino acid identities for several CryI proteins aswell as CryII, CryIII and CryIV proteins has been determined in Hodgmanand Ellar, J. DNA Sequencing and Mapping, 1, pp. 97-106 (1990).

The CryI family of ICPs contains the largest number of known toxin genesderived from B.t., as evidenced by the survey in Hofte and Whiteley(1989) and by subsequent reports of CryI-type ICPs.

Schnepf et al., J. Biol. Chem., 260, pp. 6264-6272 (1985), reported thecomplete nucleotide sequence for a toxin gene from B.t. kurstaki HD-1.This gene was subsequently classified as cryIA(a) by Hofte and Whiteley(1989). The published open reading frame extends 1176 amino acids andencodes a protein with a calculated molecular mass of 133,500 Daltons(Da). Another gene, also classified as cryIA(a), was isolated from B.t.subsp. kurstaki HD-1 Dipel® by Shibano et al., Gene 34, pp. 243-251(1985). As detailed in Table 2 of Hofte and Whiteley (1989), this geneis highly related, especially in the N terminal moiety, to cryIA(a)reported by Schnepf et al. (1985). CryIA(a) protein is broadly activeagainst Lepidoptera; Hofte and Whiteley (1989) reports that four of fivetested lepidopteran insects were sensitive to this toxin.

Other ICP genes subsequently identified as cryIA(a) that are greaterthan 99% identical to the holotype cryIA(a) gene have been identified inB. thuringiensis subspecies aizawai, (Shimizu et al., Agric. Biol.Chem., 52, pp. 1565-1573 (1988)), subspecies kurstaki, (Kondo et al.,Agric. Biol. Chem., 51, pp. 455-463 (1987)), and subspecies entomocidus(Masson et al., Nucleic Acids Res. 17, p. 446 (1989)). The cryI-typenucleotide sequence disclosed in European Patent Application PublicationNo. 0 367 474, published May 9, 1990, of Mycogen Corporation, reveals aDNA sequence related to the cryIA(a) gene and its encoded protein thatis 92% positionally identical to the holotype CryIA(a) ICP.

Wabiko et al., DNA, 5, pp. 305-314 (1986), describe the DNA sequence ofan insecticidal toxin gene from B.t. subsp. berliner 1715, subsequentlyclassified as cryIA(b) by Hofte and Whiteley (1989). The molecular massof the protein encoded is 130,615 Da and sequential deletions indicatethat the NH₂ -terminal 612 amino acid polypeptide is toxic tolepidopteran insects. Hofte et al., Eur. J. Biochem., 161, pp. 273-280(1986), describe the cloning and nucleotide sequencing of a variantcrystal protein gene from B.t. subsp. berliner 1715, subsequently alsoclassified as cryIA(b). The cloned gene produces an approximately130,000 Da protein which coincides with the mass of the major proteinobserved in the strain. The gene has an open reading frame of 3465 baseswhich would encode a protein 1155 amino acids in length having a mass of130,533 Da. Similarities of this sequence to the previously reportedsequences for the cloned crystal genes from B.t. kurstaki HD-1, B. t.kurstaki HD-73 and B.t. sotto are discussed in the Hofte et al. (1986)paper. Data identifying a minimal toxic fragment required forinsecticidal activity are also presented. The cryIA(b) gene discussed inHofte et al. (1986) differs in its deduced amino acid sequence by onlytwo amino acids from the CryIA(b) protein reported by Wabiko et al.

Other cryIA(b) genes have been disclosed in Geiser et al., Gene, 48, pp.109-118 (1986), Hefford et al., J. Biotechnol., 6, pp. 307-322 (1987),Oeda et al., Gene, 53, pp. 113-119 (1987), Kondo et al., supra,Fischhoff et al., Bio/Technology, 5, pp. 807-813 (1987) and Haider andEllar, Nucl. Acids Res., 16, p. 10927 (1988). Each of these six CryIA(b)ICPs is greater than 99% positionally identical to the holotype CryIA(b)toxin.

Adang et al., Gene, 36, pp. 289-300 (1985), report the cloning andcomplete nucleotide sequence of a crystal protein gene harbored on the75 kilobase (kb) plasmid of strain B.t. subsp. kurstaki HD-73. Therestriction map in the article identified this gene as holotype cryIA(c)under the current classification system of Hofte and Whiteley (1989).The complete sequence of the gene, spanning 3537 nucleotide base pairs(bp), coding for 1178 amino acids and potentially encoding a protein of133,330 Da, is shown in the article. Toxicity data against Manduca sextafor the protein made by the cryIA(c) gene are also presented. CryIA(c)toxins have been isolated from several strains of B.t. subsp. kenyaethat are highly related to the above-noted CryIA(c) toxin from B.t.subsp. kurstaki (greater than 99% positionally identical in deducedamino acid sequence) but whose protein products, although broadly activeagainst lepidopteran insects, nonetheless show quantitatively differenttoxicities for individual insect species (Von Tersch et al., Appl.Environ. Microbiol., 57, pp. 349-358 (1991)).

Brizzard et al., Nucleic Acids Res., 16, pp. 2723-2724 (1988), describethe nucleotide sequence of crystal protein gene cryA4 (subsequentlyclassified as cryIB by Hofte and Whiteley (1989)) isolated from B.t.subsp. thuringiensis HD-2. Hofte and Whiteley (1989) report aninsecticidal specificity of CryIB toxin for Pieris brassicae.

Honee et al., Nucleic Acids Res., 16, p. 6240 (1988), describe thecomplete DNA sequence for the BTVI crystal protein gene isolated fromB.t. subsp. entomocidus 60.5 (holotype cryIC by Hofte and Whiteley(1989)). This protein is reported to exhibit enhanced insecticidalactivities against Spodoptera species.

Sanchis et al., Mol. Microbiol., 3, pp. 229-238 (1989) report thenucleotide sequence for the N-terminal coding region (2470 nucleotides)and 5' flanking region of a gene from B.t. subsp. aizawai 7.29 nowclassified as the cryIC gene under the classification system of Hofteand Whiteley (1989). Sanchis et al. disclose similar information aboutthe cryIC gene in European Patent Application Publication No. 0 295 156,published Dec. 14, 1988. The open reading frame encodes a truncatedpolypeptide 824 amino acids long with a calculated mass of 92,906 Da.

A gene isolated from B.t. subspecies aizawai and now classified asholotype cryID under the Hofte and Whiteley (1989) system is disclosedin European Patent Application Publication No. 0 358 557, published Mar.14, 1990 of Plant Genetic Systems, N. V. Hofte and Whiteley (1989)report selective lepidopteran toxicity against Manduca sexta for theCryID protein, the CryID toxin being largely inactive against otherlepidopteran insects tested.

The holotype cryIE gene, found in a B.t. subspecies darmstadiensisstrain, is disclosed in European Patent Application Publication No. 0358 557, supra. A highly related cryIE gene from B.t. subsp. kenyae isdisclosed by Visser et al., J. Bacteriol., 172, pp. 6783-6788 (1990).

Visser, Mol. Gen. Genet., 212, pp. 219-224 (1988) report the isolationand analysis of five toxin genes belonging to four different genefamilies from B.t. entomocidus 60.5, one of which is reported by Honeeet al. (1988), supra. Two of these genes, BTIV and BTVIII, arecryIA(a)-type genes according to the Hofte and Whiteley (1989)classification scheme. The BTVI gene, also reported by Honee et al.(1988) supra, is a cryIC gene according to the Hofte and Whiteley (1989)classification scheme. The authors state that the restriction map foranother gene, designated BTV, closely resembles that identified for thecryID gene isolated from B.t. strain HD68 subsp. aizawai, and disclosedin European Patent Application Publication No. 0 358 557, supra. A fifthgene, designated BTVII, is also identified and its restriction mapdiffers significantly from the other four genes described. Toxicity dataagainst several lepidopteran insects, S. exigua, S. littoralis, H.virescens and P. brassicae, are presented for each of the isolates. TheBTV gene product was inactive against all insects tested. The BTVIprotein is highly active against Spodoptera larvae, and the BTVIIprotein is toxic to P. brassicae.

Additional genes within the cryI family have been reported in theliterature. A gene found in B.t. subsp. aizawai and described as cryIFis disclosed by Chambers et al. in J. Bacteriol., 173, pp. 3966-3976(1991) and in PCT International Publication No. WO91/16434, publishedOct. 31, 1991. A gene described as cryIG from B.t. subsp. galleria isdisclosed by Smulevitch et al., FEBS Lett., 293, pp. 25-28 (1991). Agene that is highly related to the cryIG gene has been isolated fromB.t. DSIR 517 by Gleave et al., J. Gen. Microbiol., 138, pp. 55-62(1992).

A novel gene related to cryI-type genes is disclosed in PCTInternational Publication No. WO 90/13651, published Nov. 15, 1990, ofImperial Chemical Industries PLC. This gene encodes an 81 kDapolypeptide (Cry pJH11) that is broadly insecticidal and more distantlyrelated to the family of cryI sequences than are most other reportedcryI-type sequences. Four cryI-type sequences are disclosed in EuropeanPatent Application Publication No. 0 405 810, published Jan. 2, 1991, ofMycogen Corporation. Inspection of the cryI-type sequences revealed thatone of the disclosed genes (cry 81IB2) belongs to the cryIC class, one(cry 81IB) belongs to the cryID class, and one (cry 81IA) belongs to thecryIF class. The fourth disclosed cryI sequence (cry 81IA2) appears tobelong to a new class. Two cryI sequences are disclosed in EuropeanPatent Application Publication No. 0 401 979, published Dec. 12, 1990,of Mycogen Corporation. One of the disclosed sequences (PS82A2) appearsto encode a novel gene, the other sequence (PS82RR) is highly related tothe novel sequence cry 81IA2 disclosed in European Patent ApplicationPublication No. 0 405 810.

Five novel cry sequences are disclosed in European Patent ApplicationPublication No. 0 462 721, published Dec. 27, 1991, of MycogenCorporation. These Cry proteins are reported to be nematocidal.

SUMMARY OF THE INVENTION

Briefly stated, one aspect of the present invention relates to apurified, isolated cryET4 gene having a nucleotide base sequence codingfor the amino acid sequence shown in FIG. 1 and listed in SEQ ID NO:2.

The isolated cryET4 gene has a coding region extending from nucleotidebases 99 to 3602 (including the stop codon) in the nucleotide basesequence shown in FIG. 1 and listed in SEQ ID NO:1.

The present invention also relates to the isolated CryET4 protein whichis obtainable from the cryET4 gene, and which has the amino acidsequence shown in FIG. 1 (SEQ ID NO:2), and which is insecticidal tolepidopteran insects.

Another aspect of the present invention relates to a purified, isolatedcryET5 gene having a nucleotide base sequence coding for the amino acidsequence shown in FIG. 2 and listed in SEQ ID NO:4.

The isolated cryET5 gene has a coding region extending from nucleotidebases 67 to 3756 (including the stop codon) in the nucleotide basesequence shown in FIG. 2 and listed in SEQ ID NO:3.

The present invention also relates to the isolated CryET5 protein whichis obtainable from the cryET5 gene, and which has the amino acidsequence shown in FIG. 2 (SEQ ID NO:4), and which is insecticidal tolepidopteran insects.

Additionally, the present invention relates to biologically purecultures of a Bacillus thuringiensis bacterium designated as strainEG7279 transformed with a cryET4 gene having a coding region listed inSEQ ID NO:1 and strain EG7283 transformed with a cryET5 gene having acoding region listed in SEQ ID NO:3 or mutants thereof havinginsecticidal activity against lepidopteran insects susceptible to theCryET4 and CryET5 proteins, respectively.

The invention also relates to a biologically pure culture of a Bacillusthuringiensis bacterium designated as strain EG5847 or mutants thereofhaving insecticidal activity against lepidopteran insects susceptible toB.t. strain EG5847. B.t. strain EG5847 is a wild type isolate and is theB.t. strain from which the cryET4 and cryET5 genes were isolated.

Additional aspects of the present invention relate to recombinantplasmids containing the cryET4 and cryET5 genes; bacteria transformedwith the recombinant plasmids and capable of expressing the cryET4and/or cryET5 genes; insecticide compositions comprising the proteinsand/or one or both of the transformed bacteria and/or other bacteriacontaining the CryET4 or CryET5 protein, with an agriculturallyacceptable carrier; a method of controlling lepidopteran insects usingthe insecticides; plants transformed with and capable of expressing thecryET4 and/or cryET5 genes; and hybridization probes containing thecryET4 or cryET5 gene wherein the gene or at least an oligonucleotideportion of it is labeled for such use.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 comprises FIGS. 1A through 1J and shows the nucleotide sequenceof the cryET4 gene (SEQ ID NO:1) and the deduced amino acid sequence ofthe CryET4 protein (SEQ ID NO:2).

FIG. 2 comprises FIGS. 2A through 2J and shows the nucleotide sequenceof the cryET5 gene (SEQ ID NO:3) and the deduced amino acid sequence ofthe CryET5 protein (SEQ ID NO:4).

FIG. 3 is a photograph of an ethidium bromide stained agarose gelcontaining size-fractionated plasmids of B.t. strains EG5847 and HD-1,with plasmid sizes in megadaltons (MDa) being shown. The abbreviationsin FIG. 3 are as follows: "ori" indicates the loading site and "lin"means linear DNA.

FIG. 4 is a photograph of a Coomassie blue stained gel containingsize-fractionated proteins from B.t. strains EG5847, EG7279 and EG7283,obtained by sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE).

FIG. 5 comprises FIGS. 5A and 5B and is a restriction map of therecombinant plasmids pEG291 (5A) and pEG1108 (5B), both of which containthe cloned cryET4 gene. The location and orientation of the cryET4 geneare indicated by the arrow.

FIG. 6 comprises FIGS. 6A, 6B, 6C and 6D and is a restriction map of therecombinant plasmids pEG292 (6A), pEG300 (6B), pEG1110,(6C) and pEG1111(6D). Plasmids pEG292 and pEG300 contain 5' and 3' portions of thecryET5 gene, respectively. Plasmids pEG1110 and pEG1111 contain theentire cloned cryET5 gene. The location and direction of transcriptionof the cryET5 gene are indicated by the arrows.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Two novel Bacillus thuringiensis (B.t.) toxin genes, designated cryET4(SEQ ID NO:1) and cryET5 (SEQ ID NO:3), were obtained from a novel B.t.isolate designated EG5847. Isolation of B.t. strain EG5847, isolation ofthe novel toxin genes cryET4 and cryET5, construction of Bacillus/E.coli shuttle vectors containing cryET4 (pEG1108) and cryET5 (PEG1111),and transformation of pEG1108 and pEG1111 into a B.t. host (B.t. strainEG10368) to produce recombinant B.t. strains EG7279 and EG7283expressing respectively the CryET4 (SEQ ID NO:2) and CryET5 (SEQ IDNO:4) toxin protein gene products, are described generally in theExamples.

Subcultures of B.t. strains EG5847, EG10368, EG7279 and EG7283 weredeposited in the permanent collection of the Agricultural ResearchService Culture Collection, Northern Regional Research Laboratory(NRRL), U.S. Department of Agriculture, 1815 North University Street,Peoria, Ill. 61604, U.S.A. The accession numbers and deposit dates areas follows:

    ______________________________________                                        Subculture Accession No.    Deposit Date                                      ______________________________________                                        B.t. EG5847                                                                              NRRL B-21110     June 9, 1993                                      B.t. EG10368                                                                             NRRL B-21125     July 20, 1993                                     B.t. EG7279                                                                              NRRL B-21112     June 9, 1993                                      B.t. EG7283                                                                              NRRL B-21111     June 9, 1993                                      ______________________________________                                    

These microorganism deposits were made under the provisions of the"Budapest Treaty on the International Recognition of the Deposit ofMicroorganism for the Purposes of Patent Procedure." All restrictions onthe availability to the public of these deposited microorganisms will beirrevocably removed upon issuance of a United States patent based onthis application.

The present invention is intended to cover mutants and recombinant orgenetically engineered derivatives, e.g., truncated versions, of thecryET4 gene listed in SEQ ID NO:1 and the cryET5 gene listed in SEQ IDNO:3 that yield a protein with insecticidal properties essentially thesame as those of the CryET4 protein listed in SEQ ID NO:2 and the CryET5protein listed in SEQ ID NO:4. Likewise, the present invention coversthose gene nucleotide base sequences that encode the amino acidsequences of the CryET4 protein (SEQ ID NO:2) and the CryET5 protein(SEQ ID NO:4). Variations may be made in the cryET4 and cryET5 genenucleotide base sequences shown in FIGS. 1 and 2, respectively, andlisted in SEQ ID NO:1 and SEQ ID NO:3, respectively, which do not affectthe amino acid sequence of the gene product, since the degeneracy of thegenetic code is well known to those skilled in the art. Moreover, theremay be some variations or truncations in the coding regions of thecryET4 and cryET5 nucleotide base sequences which allow expression ofthe gene and production of functionally equivalent forms of the CryET4and CryET5 insecticidal proteins. These variations or truncations, whichcan be determined without undue experimentation by those of ordinaryskill in the art with reference to the present specification, are to beconsidered within the scope of the appended claims, since they are fullyequivalent to the specifically claimed subject matter.

It has been shown that proteins of identical structure and function maybe constructed by changing the amino acid sequence, if such changes donot alter the protein secondary structure (Kaiser and Kezdy, Science,223, pp. 249-255 (1984)). Single amino acid substitutions have beenintroduced by site-directed mutagenesis at various positions of CryIA(a)toxin protein without altering the insecticidal properties of the parenttoxin (Ge et al., Proc. Natl. Acad. Sci. USA, 86, pp. 4037-4041 (1989)).The present invention includes mutants of the amino acid sequencesdisclosed herein which have an unaltered protein secondary structure or,if the structure is altered, where the mutant has retained substantiallyequivalent biological activity compared to the unaltered protein.

The cryET4 gene (SEQ ID NO:1) and cryET5 (SEQ ID NO:3) gene are alsouseful as DNA hybridization probes, for discovering similar or closelyrelated cryET4-type and cryET5-type genes in other B.t. strains. ThecryET4 gene (SEQ ID NO:1) and cryET5 gene (SEQ ID NO:3), or uniqueportions or derivatives thereof capable of hybridizing selectively to atarget nucleic acid, e.g., homologous oligonucleotides of 12 or morenucleotides, or larger portions of the genes, that contain nucleotidesequences unique to the cryET4 gene or cryET5 gene and that aredifferent from similar sized nucleotide segments in known, prior artB.t. toxin genes, can be labeled for use as hybridization probes usingconventional procedures. An exemplary label is a radioactive label.

Both the cryET4 gene (SEQ ID NO:1), its corresponding insecticidalCryET4 protein (SEQ ID NO:2) and the cryET5 gene (SEQ ID NO:2) and itscorresponding insecticidal CryET5 protein (SEQ ID NO:4) were firstidentified in B.t. strain EG5847, a novel B.t. isolate. Thecharacteristics of B.t. strain EG5847 are more fully described in theExamples.

The Bacillus strains described herein may be cultured using conventionalgrowth media and standard fermentation techniques. The B.t. strainsharboring the cryET4 gene (SEQ ID NO:1) or the cryET5 gene (SEQ IDNO:3), or both genes, may be fermented, as described in Example 1, untilthe cultured B.t. cells reach the stage of their growth cycle when theCryET4 crystal protein (SEQ ID NO:2) or the CryET5 crystal protein (SEQID NO:4) is formed. For sporogenous B.t. strains, fermentation istypically continued through the sporulation stage when the crystalprotein is formed along with spores. The B.t. fermentation culture isthen typically harvested by centrifugation, filtration or the like toseparate fermentation culture solids containing the crystal protein fromthe culture medium.

The separated fermentation solids are primarily CryET4 crystal protein(SEQ ID NO:2) or CryET5 crystal protein (SEQ ID NO:4) and B.t. spores(if a sporulating host is employed), along with some cell debris, someintact cells and residual fermentation medium solids. If desired, thecrystal protein may be separated from the other recovered solids viaconventional methods, e.g., density gradient fractionation.

The B.t. strains exemplified in this disclosure are sporulatingvarieties (spore forming or sporogenous strains) but the cryET4 gene(SEQ ID NO:1) and cryET5 gene (SEQ ID NO:3) also have utility inasporogenous Bacillus strains, i.e., strains that produce the crystalprotein without production of spores. It should be understood thatreferences to "fermentation cultures" of B.t. strains containing thecryET4 gene (SEQ ID NO:1) or the cryET5 gene (SEQ ID NO:3) in thisdisclosure are intended to cover sporulated B.t. cultures, i.e., B.t.cultures containing the CryET1 crystal protein and spores, andsporogenous Bacillus strains that have produced crystal proteins duringthe vegetative stage, as well as asporogenous Bacillus strainscontaining the cryET4 gene (SEQ ID NO:1) or cryET5 (SEQ ID NO:3) gene inwhich the culture has reached the growth stage where the crystal proteinis actually produced.

Mutants of B.t. strains harboring the cryET4 gene (SEQ ID NO:1) orcryET5 gene (SEQ ID NO:3) can be made by procedures well known in theart. For example, an asporogenous mutant can be obtained throughethylmethane sulfonate mutagenesis. Mutants can also be made usingultraviolet light and nitrosoguanidine by procedures that are well knownto those skilled in the art. References in this specification to"mutants" of wild-type or recombinant B.t. strains harboring the cryET4gene or cryET5 gene refer to those derivatives which are capable ofproducing toxin protein exhibiting insecticidal activity againstlepidopteran insects, at least equivalent to the insecticidal activityof the parent strain.

The CryET4 protein (SEQ ID NO:2) is an insecticidal compound activeagainst a large number of lepidopteran insects, particularly thosedescribed in Example 4. The CryET4 protein (SEQ ID NO:2) may be used asthe active ingredient in insecticidal formulations useful forcontrolling lepidopteran insects.

The CryET5 protein (SEQ ID NO:4) is an insecticidal compound activeagainst a large number of lepidopteran insects, particularly thosedescribed in Example 4. The CryET5 protein (SEQ ID NO:4) may be used asthe active ingredient in insecticidal formulations useful forcontrolling lepidopteran insects.

Such insecticidal formulations or compositions typically containagriculturally acceptable carriers or adjuvants in addition to theactive ingredient and are prepared and used in a manner well known tothose skilled in the art.

The CryET4 protein (SEQ ID NO:2) or CryET5 protein (SEQ ID NO:4) may beemployed in insecticidal formulations in isolated or purified form,e.g., as the crystal protein itself. Alternatively, the CryET4 protein(SEQ ID NO:2) or CryET5 protein (SEQ ID NO:4) may be present in therecovered fermentation solids, obtained from culturing of a Bacillusstrain, e.g., Bacillus thuringiensis or other microorganism hostcarrying the cryET4 gene (SEQ ID NO:1) or cryET5 gene (SEQ ID NO:3) andcapable of producing the CryET4 or CryET5 protein. The CryET4 protein orCryET5 protein is thus associated with the B.t. bacterium which producedthe protein, as an intimate mixture of crystal protein, cell debris andspores, if any, in the recovered fermentation solids. The recoveredfermentation solids containing the CryET4 or CryET5 protein may bedried, if desired, prior to incorporation in the insecticidalformulation. Genetically engineered or transformed B.t. strains or otherhost microorganisms containing a recombinant plasmid that expresses thecloned cryET4 gene (SEQ ID NO:1) or cryET5 gene (SEQ ID NO:3), obtainedby recombinant DNA procedures, may also be used. For construction ofrecombinant B.t. strains containing either the cryET4 gene or cryET5gene, B.t. var. kurstaki strain EG10368 is a preferred host, and thisB.t. strain is utilized in Example 2. B.t. strain EG10368 is acrystal-negative, toxin plasmid-free, naturally occurring mutant of B.t.strain HD73-26 (described in U.S. Pat. No. 5,080,897, issued toGonzalez, Jr. et al. on Jan. 14, 1992) that is highly transformable withrecombinant plasmids, particularly those isolated from E. coli strains,e.g., DH5α.

The formulations or compositions of this invention containing theinsecticidal CryET4 protein (SEQ ID NO:2) or CryET5 protein (SEQ IDNO:4) as the active component are applied at an insecticidally effectiveamount which will vary depending on such factors as, for example, thespecific lepidopteran insects to be controlled, the specific plant orcrop to be treated and the method of applying the insecticidally activecompositions.

The insecticide compositions are made by formulating the insecticidallyactive component with the desired agriculturally acceptable carrier. Theformulated compositions may be in the form of a dust or granularmaterial, or a suspension in oil (vegetable or mineral), or water oroil/water emulsions, or as a wettable powder, or in combination with anyother carrier material suitable for agricultural application. Suitableagricultural carriers can be solid or liquid and are well known in theart. The term "agriculturally acceptable carrier" covers all adjuvants,e.g., inert components, dispersants, surfactants, tackifiers, binders,etc. that are ordinarily used in insecticide formulation technology;these are well known to those skilled in insecticide formulation.

The formulations containing the CryET4 protein (SEQ ID NO:2) or CryET5protein (SEQ ID NO:4) and one or more solid or liquid adjuvants areprepared in known manners, e.g., by homogeneously mixing, blendingand/or grinding the insecticidally active CryET4 or CryET5 proteincomponent with suitable adjuvants using conventional formulationtechniques.

The insecticidal compositions of this invention are applied to theenvironment of the target lepidopteran insect, typically onto thefoliage of the plant or crop to be protected, by conventional methods,preferably by spraying. Other application techniques, e.g., dusting,sprinkling, soaking, soil injection, seed coating, seedling coating orspraying, or the like, are also feasible and may be required for insectsthat cause root or stalk infestation. These application procedures arewell known in the art.

The cryET4 gene (SEQ ID NO:1) or cryET5 gene (SEQ ID NO:3) may beintroduced into a variety of microorganism hosts without undueexperimentation, using procedures well known to those skilled in the artfor transforming suitable hosts under conditions which allow for stablemaintenance and expression of the cloned genes. Maniatis et al.,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y. (1982). Suitable hosts that allow thecryET4 gene (SEQ ID NO:1) or cryET5 gene (SEQ ID NO:3) gene to beexpressed and the CryET4 protein (SEQ ID NO:2) or CryET5 protein (SEQ IDNO:4) to be produced include B.t. and other Bacillus species such as B.subtilis or B. megaterium. A general method for the transformation ofBacillus strains is provided by Macaluso et al. in J. Bacteriol., 173,pp. 1353-1356 (1991) and Mettus et al. in Appl. Environ. Microbiol., 56,pp. 1128-1134 (1990). Genetically altered or engineered microorganismscontaining the cryET4 gene (SEQ ID NO:1) or cryET5 gene (SEQ ID NO:3)can also contain other toxin genes present in the same microorganism;these genes could concurrently produce ICPs different from the CryET4protein or CryET5 protein.

Plant-colonizing or root-colonizing microorganisms may also be employedas the host for the cryET4 gene (SEQ ID NO:1) or cryET5 gene (SEQ IDNO:3). Exemplary microorganism hosts for B.t. toxin genes include theplant-colonizing microbe Clavibacter xyli subsp. cynodontis, asdescribed by Turner et al. in Appl. Environ. Microbiol., 57, pp.3522-3528, and root-colonizing pseudomonad strains, as described byObukowicz et al. in Gene, 45, pp. 327-331 (1986). Procedures such asthose described by Turner et al. (1991) supra, and Obukowicz et al.(1986), supra, are well known to those skilled in the art and availablefor introducing the cryET4 gene (SEQ ID NO:1) or cryET5 gene (SEQ IDNO:3) into such microorganism hosts under conditions which allow forstable maintenance and expression of the gene in the resultingtransformants.

The transformants, i.e., host microorganisms that harbor a cloned genein a recombinant plasmid, can be isolated in accordance withconventional methods, usually employing a selection technique, whichallows growth of only those host microorganisms that contain arecombinant plasmid. The transformants then can be tested forinsecticidal activity. These techniques are standard procedures wellknown to those skilled in the art.

Characteristics of particular interest in selecting a host cell forpurposes of production include ease of introducing the gene into thehost, availability of expression systems, efficiency of expression,stability of the CryET4 or CryET5 insecticidal protein in the host, andthe presence of auxiliary genetic capabilities. The cellular hostcontaining the insecticidal cryET4 gene (SEQ ID NO:1) or cryET5 gene(SEQ ID NO:3) may be grown in any convenient nutrient medium, whereexpression of the cryET4 gene or cryET5 gene is obtained and CryET4protein (SEQ ID NO:2) or CryET5 protein (SEQ ID NO:4) produced,typically to sporulation. The sporulated cells containing the crystalprotein may then be harvested in accordance with conventional methods,e.g., centrifugation or filtration.

The cryET4 gene (SEQ ID NO:1) or cryET5 gene (SEQ ID NO:3), particularlythe toxin portion (N-terminal moiety) thereof, may also be incorporatedinto a plant which is capable of expressing the gene and producingCryET4 protein (SEQ ID NO:2) or CryET5 protein (SEQ ID NO:4), renderingthe plant more resistant to insect attack. Genetic engineering of plantswith the cryET4 gene (SEQ ID NO:1) or cryET5 gene (SEQ ID NO:3) may beaccomplished by introducing the desired DNA containing the gene intoplant tissues or cells, using DNA molecules of a variety of forms andorigins that are well known to those skilled in plant geneticengineering. Examples of techniques for introducing DNA into planttissue are disclosed in European Patent Application Publication No. 0289 479, published Nov. 1, 1988, of Monsanto Company and by Perlak etal. in "Modification of the Coding Sequence Enhances Plant Expression ofInsect Control Protein Genes," Proc. Natl. Acad. Sci. USA, 88, pp.3324-3328 (1991).

DNA containing the cryET4 gene (SEQ ID NO:1) or cryET5 gene (SEQ IDNO:3) or a modified gene, operatively associated with a suitable plantpromoter, e.g., CaMV35S, capable of effecting production of the CryET4protein (SEQ ID NO:2) or CryET5 protein (SEQ ID NO:4), may be deliveredinto the plant cells or tissues directly by infectious plasmids, such asTi, the plasmid from Agrobacterium tumefaciens, viruses ormicroorganisms like A. tumefaciens. Additionally, the use of lysosomesor liposomes, microinjection by mechanical methods and by othertechniques familiar to those skilled in plant genetic engineering may beused.

The basic methods employed in the construction and evaluation of therecombinant plasmids and recombinant microorganism hosts described inthis specification are generally well know to those proficient in theart of molecular cloning. Descriptions of these general laboratoryprocedures and definitions of nomenclature may be found in Maniatis etal., Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (1982) and in a subsequentedition by Sambrook et al. (1989).

The characteristics of the CryET4 protein (SEQ ID NO:2) and CryET5protein (SEQ ID NO:4), sequencing of the cryET4 gene (SEQ ID NO:1) andcryET5 gene (SEQ ID NO:3), comparison of sequence data to known B.t.toxin genes and insecticidal activity of the CryET4 and CryET5 proteinsare described in the following specific, non-limiting examples.

EXAMPLE 1 Characterization of B.t. EG5847

B.t. strain EG5847 is a wild-type isolate, identified by visualexamination of the colony as exhibiting a unique crystal morphology, andwas isolated as a colony from maize dust. The colony containedendospores and bipyramidal and flat, diamond-shaped crystallineinclusions. Subsequent insect bioassay of this wild-type B.t. strainconfirmed its insecticidal activity towards lepidopteran insects.

The complement of native plasmids contained within isolated B.t. EG5847was determined by modified Eckhardt agarose gel electrophoresis asdescribed by Gonzalez, Jr. et al., in Proc. Natl. Acad. Sci. USA, 79,pp. 6951-6955 (1982). The results, as shown in FIG. 3, revealed thepresence of 5, 8, 12, 18 and 110 MDa plasmids. This pattern of nativeplasmids did not correspond to patterns typical of known serovars(Carlton and Gonzalez, pp. 246-252, in Molecular Biology of MicrobialDifferentiation, J. A. Hoch and P. Setlow, ed., American Society forMicrobiology, Washington, D.C. (1985)).

Wild-type B.t. strain EG5847 was grown for five days at 25° C. in DSMmedium (described by Donovan et al. in Appl. Environm. Microbiol., 58,pp. 3921-3927 (1992)) until sporulation and cell lysis had occurred.Recombinant B.t. strains EG7279 (Example 2), containing the cryET4 gene,and EG7283 (Example 2), containing the cryET5 gene, were grown in DSMmedium containing 3 μg of chloramphenicol per ml in a similar manner.Fermentation solids containing spores and crystal proteins were isolatedby centrifugation. Crystal proteins were purified from the spores andcell debris by sucrose density gradient centrifugation (described byKoller et al. in Biochem. Biophys. Res. Communic., 184, pp. 692-699(1992)). Aliquots of the washed crystals were solubilized by heating inLaemmli buffer (10% (w/w) glycerol, 5% (w/w) 2-mercaptoethanol, 1% (w/v)SDS, 0.188 M Tris HCl pH 6.8, 0.01% (v/v) bromphenol blue) at 100° C.for 5 minutes. The solubilized crystal proteins were size fractionatedby SDS-polyacrylamide gel electrophoresis. After size fractionation, theproteins were visualized by staining with Coomassie Blue R-250 dye.

FIG. 4 shows the results of these protein size fractionation analyseswhere lane 1 is a molecular mass size standard, lane 2 is B.t. strainEG5847, lane 3 is B.t. strain EG2729 and lane 4 is B.t. strain EG7283.The numbers on the left side indicate the apparent molecular masses, inkilodaltons (kDa), of the crystal proteins synthesized by the B.t.strains. As shown in lane 3, a crystal protein having a mass ofapproximately 130 kDa was observed from EG7279. As shown in lane 4 forEG7283, a crystal protein having a mass of approximately 130 kDa wasproduced. The wild type strain EG5847 exhibited a large protein band ofapproximately 130 kDa. The observed masses of the crystal proteins arein agreement with the masses predicted from the DNA sequences obtainedin Example 3.

EXAMPLE 2 Cloning of the cryET4 and cryET5 Genes

Genomic DNA was isolated from B.t. strain EG5847 and then digested withHindIII. cryI-like genes were identified by Southern blotting (describedby Southern, J. Mol. Biol. 98, pp. 503-517 (1975)). A radiolabelled 700bp EcoR1 fragment of the cryIA(a) gene (described by Schnepf et al., J.Biol. Chem., 260, pp. 6264-6272 (1985)) was used as a hybridizationprobe to identify cryI-like genes containing HindIII restrictionfragments of EG5847 DNA. The 700 bp cryIA(a) fragment hybridized toseveral HindIII restriction fragments of EG5847 DNA including fragmentsof approximately 5.0 kb and 4.7 kb.

The 5.0 kb and 4.7 kb cryIA(a)-hybridizing HindIII restriction fragmentsof B.t. strain EG5847 were cloned as follows. DNA fragments ofapproximately 4-8 kb from HindIII digests of EG5847 genomic DNA werepurified by agarose gel electrophoresis and electroelution. Thesefragments were ligated to the E. coli plasmid vector pUC18 and theligation mixture was then used to transform E. coli.Ampicillin-resistant E. coli colonies were blotted to nitrocellulosefilters (Grunstein et al., Proc. Natl. Acad. Sci. USA 72, pp. 3961-3965(1975)). The filters were probed with the radiolabelled 700 bp cryIA(a)gene fragment. Two positive colonies, designated as E. coli strainsEG7286 and EG7287, were identified and were selected for furtheranalysis.

HindIII digestion of a plasmid, designated pEG291, isolated from E. colistrain EG7286 revealed a HindIII insert fragment 5.0 kb in size inpUC18. The restriction map of plasmid pEG291 is shown in FIG. 5A.

An E. coli/B. thuringiensis shuttle vector containing the 5.0 kb HindIIIfragment was constructed by ligating BamHI digested Bacillus plasmidpNN101 (Norton et al., Plasmid, 13, pp. 211-214 (1985)) into the uniqueBamHI site of pEG291 (FIG. 5B). The resulting plasmid, designatedpEG1108 (FIG. 5B), contains a full length open reading frame which hasbeen designated as the cryET4 gene (SEQ ID NO:1).

E. coli strain EG7287 contained a plasmid, designated pEG292, which hada 4.7 kb HindIII insert in pUC18. DNA sequencing as described in Example3 indicated that an open reading frame present in the 4.7 kb insert wastruncated at its 3'-end (FIG. 6A). The truncated portion was isolatedusing a synthetic oligonucleotide having the sequence5'-AAGTTTCGCATCCATCGATG-3' (SEQ ID NO:5). The oligonucleotide,designated WD162, was homologous to nucleotides 2253 to 2272 of the openreading frame identified in plasmid pEG292. Southern blot analyses asdescribed above indicated that WD162 (SEQ ID NO:5) hybridized to a 3.2kb HincII restriction fragment of EG5847 DNA. Radiolabelled WD162 wasthen used in colony blot experiments as described above to probe E. colicells that contained a plasmid library consisting of size-selectedHincII restriction fragments of EG5847 DNA. Several E. coli colonieshybridized with WD162 and one colony, designated E. coli strain EG7288,was selected for further analysis.

HincII restriction analysis of a recombinant plasmid, designated pEG300,isolated from E. coli EG7288, indicated that a 3.9 kb HincII fragmentwas present in pUC18. DNA sequencing as described below showed thatpEG300 contained an open reading frame truncated at its 5'-end by theHincII cleavage site (FIG. 6B).

The full length open reading frame was constructed by excising a 2.6 kbXbaI-BsmI fragment containing the 5' portion of the open reading framefrom plasmid pEG292 and inserting the fragment into the XbaI-BsmIrestriction sites of plasmid pEG300 (FIG. 6A and 6B). The resultingplasmid, designated pEG1110 (FIG. 6C), contains a full length openreading frame which has been designated as the cryET5 gene (SEQ IDNO:3).

An E. coli/B. thuringiensis expression vector containing the full lengthopen reading frame of the cryET5 gene was constructed by ligating XbaIdigested Bacillus plasmid pNN101 (Norton, supra) into the unique XbaIsite of plasmid pEG1110 (FIG. 6D). The resulting construct wasdesignated plasmid pEG1111.

Plasmids pEG1108 and pEG1111 are capable of replicating in both E. coliand B.t. The plasmids were transformed by electroporation (Macaluso etal., J. Bacteriol., 173, pp. 1353-1356 (1991)) into the acrystalliferousB.t. strain EG10368 resulting in B.t. strains EG7279(pEG1108) andEG7283(pEG1111), respectively containing the cryET4 and cryET5 genes.Both of these B.t. strains are capable of expressing their respectiveprotein toxin genes, as described in Example 4.

EXAMPLE 3 Sequencing of the cryET4 and cryET5 Genes

The complete DNA sequence of the cryET4 gene was determined usingplasmid pEG291 (Example 2). Plasmid pEG291 was sequenced by standardmethods (Sanger et al., Proc. Natl. Acad. Sci. USA, 74, pp. 5463-5467(1977)). The DNA sequences of the appropriate subclones of the 5.0 kbHindIII fragment were joined to give a continuous sequence of 3713nucleotides which is shown in FIG. 1 and is designated as SEQ ID NO:1.Inspection of the sequence revealed an open reading frame beginning atposition 99 and extending to position 3602 (including the stop codon).The gene has been designated cryET4. The deduced 1167 amino acidsequence of the gene product is shown in FIG. 1 and is designated as SEQID NO:2. The mass of the CryET4 protein (SEQ ID NO:2) encoded by thecryET4 gene (SEQ ID NO:1), as deduced from the open reading frame, is132,774 Da. Among CryI-type protein toxins reported in the literature,the CryIA(a) protein appears to be most closely related to the CryET4protein. CryET4 exhibits 69% amino acid homology with CryIA(a).

The complete DNA sequence of the cryET5 gene (SEQ ID NO:3) wasdetermined by the Sanger method as described above. Subcloned genefragments from pEG292, pEG300 and pEG1110 were sequenced. The DNAsequences of the subcloned fragments were joined to give a continuoussequence of 3,934 nucleotides which is shown in FIG. 2 and is designatedas SEQ ID NO:3. Inspection of the sequence revealed an open readingframe beginning at position 67 and extending to position 3756 (includingthe stop codon). The gene has been designated cryET5. The deduced 1229amino acid sequence of the gene product encoded by the cryET5 gene (SEQID NO:3) is shown in FIG. 2 and is designated as SEQ ID NO:4. The massof the CryET5 protein (SEQ ID NO:4) encoded by the cryET5 gene (SEQ IDNO:3), as deduced from the open reading frame, is 139,783 Da. AmongCryI-type proteins reported in the literature, the CryIB protein appearsto be most closely related to the CryET5 protein. CryET5 exhibits 83%amino acid homology with CryIB.

EXAMPLE 4 Insecticidal Activity of Recombinant Strains Harboring cryET4and cryET5 Genes

PLC₅₀ values of purified CryET4 and CryET5 crystal proteins weredetermined against lepidopteran insects, and these are listed in Tables1 and 2, respectively. The PLC₅₀ dose is that amount of insecticidalcrystal protein (ICP) which killed half of the insects tested, i.e., themedian lethal concentration. CryET4 and CryET5 crystal proteins wereisolated from B.t. strains EG7279 and EG7283, respectively, (describedin Example 2) by sucrose density gradient centrifugation as describedabove. The amount of crystal protein recovered from the gradients wasquantified by the Bradford protein assay (Bradford, Anal. Biochem., 72,p. 248 (1976)) after solubilization of the recovered crystal proteinswith base and a reducing agent. Known amounts of purified crystals werediluted in 0.005% Triton® X-100 (v/v). Aliquots of eight two-fold serialdilutions (50 μl) were applied to the surfaces of 32 wells (1.8 cm²surface area) containing insect diet and dried for 1 hour at 30° C. Ageneral purpose Noctuidae artificial diet (E. G. King et al., Handbookof Insect Rearing, Vol. 2, P. Singh and R. F. Moore (eds.), pp. 323-328,Elsevier Science Publishers B. V., Amsterdam (1985)) was used forTrichoplusia ni, Ostrinia nubilalis and Heliothis virescens. Otherstandard diets were used for the other lepidopteran insects tested. Oneneonate larva (third-instar larva in the case of P. xylostella) wasadded to each well, and the wells were incubated at 30° C. Mortality wasrecorded after seven days.

The insecticidal activity of CryET4 protein was compared with theactivity of CryIA(a) protein (Schnepf et al., J. Biol. Chem., 260, pp.6264-6272 (1985)). CryET4 exhibits a 69% amino acid sequence homologywith CryIA(a). The results are presented in Table 1.

                  TABLE 1                                                         ______________________________________                                        PLC.sub.50 Bioassay Activity of Purified CryET4                                          PLC.sub.50 (ng ICP/well)                                           Insect Species                                                                             CryET4        CryIA(a)                                           ______________________________________                                        Heliothis virescens                                                                        593 (493-711)**                                                                             94 (76-113)                                        Helicoverpa zea                                                                            1,290 (1,046-1,599)                                                                         3,725 (3,004-4,551)                                Lymantria dispar                                                                           9,929 (5,767-26,039)                                                                        185 (138-243)                                      Ostrinia nubilalis                                                                         197 (121-299) 34 (27-42)                                         Pseudoplusia includens                                                                     33 (29-37)    14 (12-16)                                         Plutella xylosella                                                                         30 (22-41)    12 (10-14)                                         Javelin ®*-resistant                                                                   4,758 (3,135-6,697)                                                                         >50,000                                            P. xylostella                                                                 Spodoptera exiqua                                                                          1,748 (1,286-2,591)                                                                         >20,000                                            Spodoptera frugiperda                                                                      1,161 (555-2,115)                                                                           >10,000                                            Trichoplusia ni                                                                            62 (53-74)    80 (54-113)                                        ______________________________________                                         *Javelin is a commercial B.t. bioinsecticide.                                 **Range in parentheses indicates 95% confidence level.                   

The PLC₅₀ results in Table 1 indicate that the CryET4 protein (SEQ IDNO:2) exhibits good insecticidal activity to a broad spectrum oflepidopteran insects.

The results show that the CryET4 protein is more toxic than CryIA(a)against Helicoverpa zea (corn earworm/bollworm), Javelin®-resistantPlutella xylostella (diamondback moth), Spodoptera exigua (beetarmyworm) and Spodoptera frugiperda (fall armyworm).

Particularly noteworthy is the very good activity against Spodopteraexigua (beet armyworm), an insect pest that not only is not susceptibleto CryIA(a), but also is recalcitrant to most B.t. toxin proteins, andvery good activity against Spodoptera frugiperda (fall armyworm),another recalcitrant insect pest. Activity against Pseudoplusiaincludens (soybean looper), Plutella xylostella (diamondback moth) andTrichoplusia ni (cabbage looper) was also good, comparable to thatexhibited by CryIA(a).

Insect bioassay tests with CryET4 protein were also carried out againstanother lepidopteran insect, Agrotis ipsilon (black cutworm), which wasfound not to be susceptible to control with CryET4.

The insecticidal activity of CryET5 protein was compared with theactivity of CryIB protein (Brizzard et al., Nucleic Acids Res. 16,2723-2724 (1988)). CryET5 exhibits an 83% amino acid sequence homologywith CryIB. Dilutions of purified CryET5 crystals were prepared in0.005% Triton® X-100. Aliquots of appropriate dilutions (50 μl) wereapplied to the surfaces of 32 wells and assayed as indicated above. Theresults are presented in Table 2.

                  TABLE 2                                                         ______________________________________                                        PLC.sub.50 Bioassay Activity of Purified CryET5                                          PLC.sub.50 (ng ICP/well)                                           Insect Species                                                                             CryET5        CryIB                                              ______________________________________                                        Lymantria dispar                                                                           880 (555-1,397)**                                                                           3,580 (1,293-20,123)                               Ostrinia nubilalis                                                                         32 (29-37)    83 (51-123)                                        Pseudoplusia includens                                                                     555 (437-646) 52 (44-61)                                         Piutella xylostella                                                                        157 (127-193) 27 (23-32)                                         Javelin ®*-resistant                                                      P. xylostella                                                                              47 (23-80)    43 (35-55)                                         Spodoptera frugiperda                                                                      2,612 (1,831-4,514)                                                                         >10,000                                            Tricnoplusia ni                                                                            22 (19-27)    205 (176-241)                                      ______________________________________                                         *Javelin is a commercial B.t. bioinsecticide.                                 **Range in parentheses indicates 95% confidence level.                   

The PLC₅₀ results in Table 2 indicate that the CryET5 protein (SEQ IDNO:4) exhibits good insecticidal activity to a broad spectrum oflepidopteran insects.

The results show that the CryET5 protein is more toxic than CryIBagainst Spodoptera frugiperda (fall armyworm) and Trichoplusia ni(cabbage looper). The CryET5 protein and CryIB protein both exhibitedexcellent insecticidal activity against Javelin®-resistant Flutellaxylostella (diamondback moth), a B.t.-resistant insect pest that is notsusceptible to CryIA-type toxin proteins, and to Ostrinia nubilalis(European corn borer).

Insect bioassay tests with CryET5 protein were also carried out againsta few other lepidopteran insects, but these were found not to besusceptible to control with CryET5: Agrotis ipsilon (black cutworm),Heliothis virescens (tobacco budworm), Helicoverpa zea (cornearworm/bollworm) and Spodoptera exigua (beet armyworm).

The present invention may be embodied in other specific forms withoutdeparting from the spirit or essential attributes thereof and,accordingly, reference should be made to the appended claims, ratherthan to the foregoing specification as indicating the scope of theinvention.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                - (1) GENERAL INFORMATION:                                                    -    (iii) NUMBER OF SEQUENCES: 5                                             - (2) INFORMATION FOR SEQ ID NO:1:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 3713 base                                                         (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: circular                                              -     (ii) MOLECULE TYPE: DNA (genomic)                                       -     (ix) FEATURE:                                                                     (A) NAME/KEY: CDS                                                             (B) LOCATION: 99..3602                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                 - AAATTCATAA TATGAATCAT ACGTTTTAAA GTGTTGTGAA GAAAAGAGAA TT - #GATCTTTA         60                                                                          #AAT AAT        113ACCA AAGAGAAAGG GGTAACTT ATG GAG ATA                       #      Met Glu Ile Asn Asn                                                    #     5  1                                                                    - CAG AAG CAA TGC ATA CCA TAT AAT TGC TTA AG - #T AAT CCT GAG GAA GTA          161                                                                          Gln Lys Gln Cys Ile Pro Tyr Asn Cys Leu Se - #r Asn Pro Glu Glu Val           #                 20                                                          - CTT TTG GAT GGG GAG AGG ATA TTA CCT GAT AT - #C GAT CCA CTC GAA GTT          209                                                                          Leu Leu Asp Gly Glu Arg Ile Leu Pro Asp Il - #e Asp Pro Leu Glu Val           #             35                                                              - TCT TTG TCG CTT TTG CAA TTT CTT TTG AAT AA - #C TTT GTT CCA GGG GGA          257                                                                          Ser Leu Ser Leu Leu Gln Phe Leu Leu Asn As - #n Phe Val Pro Gly Gly           #         50                                                                  - GGC TTT ATT TCA GGA TTA GTT GAT AAA ATA TG - #G GGG GCT TTG AGA CCA          305                                                                          Gly Phe Ile Ser Gly Leu Val Asp Lys Ile Tr - #p Gly Ala Leu Arg Pro           #     65                                                                      - TCT GAA TGG GAC TTA TTT CTT GCA CAG ATT GA - #A CGG TTG ATT GAT CAA          353                                                                          Ser Glu Trp Asp Leu Phe Leu Ala Gln Ile Gl - #u Arg Leu Ile Asp Gln           # 85                                                                          - AGA ATA GAA GCA ACA GTA AGA GCA AAA GCA AT - #C ACT GAA TTA GAA GGA          401                                                                          Arg Ile Glu Ala Thr Val Arg Ala Lys Ala Il - #e Thr Glu Leu Glu Gly           #                100                                                          - TTA GGG AGA AAT TAT CAA ATA TAC GCT GAA GC - #A TTT AAA GAA TGG GAA          449                                                                          Leu Gly Arg Asn Tyr Gln Ile Tyr Ala Glu Al - #a Phe Lys Glu Trp Glu           #           115                                                               - TCA GAT CCT GAT AAC GAA GCG GCT AAA AGT AG - #A GTA ATT GAT CGC TTT          497                                                                          Ser Asp Pro Asp Asn Glu Ala Ala Lys Ser Ar - #g Val Ile Asp Arg Phe           #       130                                                                   - CGT ATA CTT GAT GGT CTA ATT GAA GCA AAT AT - #C CCT TCA TTT CGG ATA          545                                                                          Arg Ile Leu Asp Gly Leu Ile Glu Ala Asn Il - #e Pro Ser Phe Arg Ile           #   145                                                                       - ATT GGA TTT GAA GTG CCA CTT TTA TCG GTT TA - #T GTT CAA GCA GCT AAT          593                                                                          Ile Gly Phe Glu Val Pro Leu Leu Ser Val Ty - #r Val Gln Ala Ala Asn           150                 1 - #55                 1 - #60                 1 -       #65                                                                           - CTA CAT CTC GCT CTA TTG AGA GAT TCT GTT AT - #T TTT GGA GAG AGA TGG          641                                                                          Leu His Leu Ala Leu Leu Arg Asp Ser Val Il - #e Phe Gly Glu Arg Trp           #               180                                                           - GGA TTG ACG ACA AAA AAT GTC AAT GAT ATC TA - #T AAT AGA CAA ATT AGA          689                                                                          Gly Leu Thr Thr Lys Asn Val Asn Asp Ile Ty - #r Asn Arg Gln Ile Arg           #           195                                                               - GAA ATT CAT GAA TAT AGC AAT CAT TGC GTA GA - #T ACG TAT AAC ACA GAA          737                                                                          Glu Ile His Glu Tyr Ser Asn His Cys Val As - #p Thr Tyr Asn Thr Glu           #       210                                                                   - CTA GAA CGT CTA GGG TTT AGA TCT ATA GCG CA - #G TGG AGA ATA TAT AAT          785                                                                          Leu Glu Arg Leu Gly Phe Arg Ser Ile Ala Gl - #n Trp Arg Ile Tyr Asn           #   225                                                                       - CAG TTT AGA AGA GAA CTA ACA CTA ACT GTA TT - #A GAT ATT GTC GCT CTT          833                                                                          Gln Phe Arg Arg Glu Leu Thr Leu Thr Val Le - #u Asp Ile Val Ala Leu           230                 2 - #35                 2 - #40                 2 -       #45                                                                           - TTC CCG AAC TAT GAC AGT AGA CTG TAT CCG AT - #C CAA ACT TTT TCT CAA          881                                                                          Phe Pro Asn Tyr Asp Ser Arg Leu Tyr Pro Il - #e Gln Thr Phe Ser Gln           #               260                                                           - TTG ACA AGA GAA ATT GTT ACA TCC CCA GTA AG - #C GAA TTT TAT TAT GGT          929                                                                          Leu Thr Arg Glu Ile Val Thr Ser Pro Val Se - #r Glu Phe Tyr Tyr Gly           #           275                                                               - GTT ATT AAT AGT GGT AAT ATA ATT GGT ACT CT - #T ACT GAA CAG CAG ATA          977                                                                          Val Ile Asn Ser Gly Asn Ile Ile Gly Thr Le - #u Thr Glu Gln Gln Ile           #       290                                                                   - AGG CGA CCA CAT CTT ATG GAC TTC TTT AAC TC - #C ATG ATC ATG TAT ACA         1025                                                                          Arg Arg Pro His Leu Met Asp Phe Phe Asn Se - #r Met Ile Met Tyr Thr           #   305                                                                       - TCA GAT AAT AGA CGG GAA CAT TAT TGG TCA GG - #A CTT GAA ATG ACG GCT         1073                                                                          Ser Asp Asn Arg Arg Glu His Tyr Trp Ser Gl - #y Leu Glu Met Thr Ala           310                 3 - #15                 3 - #20                 3 -       #25                                                                           - TAT TTT ACA GGA TTT GCA GGA GCT CAA GTG TC - #A TTC CCT TTA GTC GGG         1121                                                                          Tyr Phe Thr Gly Phe Ala Gly Ala Gln Val Se - #r Phe Pro Leu Val Gly           #               340                                                           - ACT AGA GGG GAG TCA GCT CCA CCA TTA ACT GT - #T AGA AGT GTT AAT GAT         1169                                                                          Thr Arg Gly Glu Ser Ala Pro Pro Leu Thr Va - #l Arg Ser Val Asn Asp           #           355                                                               - GGA ATT TAT AGA ATA TTA TCG GCA CCG TTT TA - #T TCA GCG CCT TTT CTA         1217                                                                          Gly Ile Tyr Arg Ile Leu Ser Ala Pro Phe Ty - #r Ser Ala Pro Phe Leu           #       370                                                                   - GGC ACC ATT GTA TTG GGA AGT CGT GGA GAA AA - #A TTT GAT TTT GCG CTT         1265                                                                          Gly Thr Ile Val Leu Gly Ser Arg Gly Glu Ly - #s Phe Asp Phe Ala Leu           #   385                                                                       - AAT AAT ATT TCA CCT CCG CCA TCT ACA ATA TA - #C AGA CAT CCT GGA ACA         1313                                                                          Asn Asn Ile Ser Pro Pro Pro Ser Thr Ile Ty - #r Arg His Pro Gly Thr           390                 3 - #95                 4 - #00                 4 -       #05                                                                           - GTA GAT TCA CTA GTC AGT ATA CCG CCA CAG GA - #T AAT AGC GTA CCA CCG         1361                                                                          Val Asp Ser Leu Val Ser Ile Pro Pro Gln As - #p Asn Ser Val Pro Pro           #               420                                                           - CAC AGG GGA TCT AGT CAT CGA TTA AGT CAT GT - #T ACA ATG CGC GCA AGT         1409                                                                          His Arg Gly Ser Ser His Arg Leu Ser His Va - #l Thr Met Arg Ala Ser           #           435                                                               - TCC CCT ATA TTC CAT TGG ACG CAT CGC AGC GC - #A ACC ACT ACA AAT ACA         1457                                                                          Ser Pro Ile Phe His Trp Thr His Arg Ser Al - #a Thr Thr Thr Asn Thr           #       450                                                                   - ATT AAT CCA AAT GCT ATT ATC CAA ATA CCA CT - #A GTA AAA GCA TTT AAC         1505                                                                          Ile Asn Pro Asn Ala Ile Ile Gln Ile Pro Le - #u Val Lys Ala Phe Asn           #   465                                                                       - CTT CAT TCA GGT GCC ACT GTT GTT AGA GGA CC - #A GGG TTT ACA GGT GGT         1553                                                                          Leu His Ser Gly Ala Thr Val Val Arg Gly Pr - #o Gly Phe Thr Gly Gly           470                 4 - #75                 4 - #80                 4 -       #85                                                                           - GAT ATC CTT CGA AGA ACG AAT ACT GGC ACA TT - #T GCA GAT ATG AGA GTA         1601                                                                          Asp Ile Leu Arg Arg Thr Asn Thr Gly Thr Ph - #e Ala Asp Met Arg Val           #               500                                                           - AAT ATT ACT GGG CCA TTA TCC CAA AGA TAT CG - #T GTA AGA ATT CGC TAT         1649                                                                          Asn Ile Thr Gly Pro Leu Ser Gln Arg Tyr Ar - #g Val Arg Ile Arg Tyr           #           515                                                               - GCT TCT ACG ACA GAT TTA CAA TTT TTC ACG AG - #A ATC AAT GGA ACT TCT         1697                                                                          Ala Ser Thr Thr Asp Leu Gln Phe Phe Thr Ar - #g Ile Asn Gly Thr Ser           #       530                                                                   - GTA AAT CAA GGT AAT TTC CAA AGA ACT ATG AA - #T AGA GGG GAT AAT TTA         1745                                                                          Val Asn Gln Gly Asn Phe Gln Arg Thr Met As - #n Arg Gly Asp Asn Leu           #   545                                                                       - GAA TCT GGA AAC TTT AGG ACT GCA GGA TTT AG - #T ACG CCT TTT AGT TTT         1793                                                                          Glu Ser Gly Asn Phe Arg Thr Ala Gly Phe Se - #r Thr Pro Phe Ser Phe           550                 5 - #55                 5 - #60                 5 -       #65                                                                           - TCA AAT GCG CAA AGT ACA TTC ACA TTG GGT AC - #T CAG GCT TTT TCA AAT         1841                                                                          Ser Asn Ala Gln Ser Thr Phe Thr Leu Gly Th - #r Gln Ala Phe Ser Asn           #               580                                                           - CAG GAA GTT TAT ATA GAT CGA ATT GAA TTT GT - #C CCG GCA GAA GTA ACA         1889                                                                          Gln Glu Val Tyr Ile Asp Arg Ile Glu Phe Va - #l Pro Ala Glu Val Thr           #           595                                                               - TTC GAG GCA GAA TCT GAT TTA GAA AGA GCG CA - #A AAG GCG GTG AAT GCC         1937                                                                          Phe Glu Ala Glu Ser Asp Leu Glu Arg Ala Gl - #n Lys Ala Val Asn Ala           #       610                                                                   - CTG TTT ACT TCT ACA AAC CAA CTA GGG CTA AA - #A ACA GAT GTG ACG GAT         1985                                                                          Leu Phe Thr Ser Thr Asn Gln Leu Gly Leu Ly - #s Thr Asp Val Thr Asp           #   625                                                                       - TAT CAG ATT GAT CAA GTG TCC AAT TTA GTA GA - #A TGT TTA TCA GAT GAA         2033                                                                          Tyr Gln Ile Asp Gln Val Ser Asn Leu Val Gl - #u Cys Leu Ser Asp Glu           630                 6 - #35                 6 - #40                 6 -       #45                                                                           - TTT TGT CTG GAT GAA AAG AGA GAA TTG TCC GA - #G AAA GTC AAA CAT GCA         2081                                                                          Phe Cys Leu Asp Glu Lys Arg Glu Leu Ser Gl - #u Lys Val Lys His Ala           #               660                                                           - AAG CGA CTT AGT GAT AAG CGG AAC CTA CTT CA - #A GAT CCA AAC TTC ACA         2129                                                                          Lys Arg Leu Ser Asp Lys Arg Asn Leu Leu Gl - #n Asp Pro Asn Phe Thr           #           675                                                               - TCT ATC AAT AGA CAA CTA GAC CGT GGA TGG AG - #A GGA AGT ACG GAT ATT         2177                                                                          Ser Ile Asn Arg Gln Leu Asp Arg Gly Trp Ar - #g Gly Ser Thr Asp Ile           #       690                                                                   - ACC ATC CAA GGA GGA AAT GAC GTA TTC AAA GA - #G AAT TAC GTC ACA CTA         2225                                                                          Thr Ile Gln Gly Gly Asn Asp Val Phe Lys Gl - #u Asn Tyr Val Thr Leu           #   705                                                                       - CCA GGT ACC TTT GAT GAG TGT TAT CCA ACG TA - #T TTG TAT CAA AAA ATA         2273                                                                          Pro Gly Thr Phe Asp Glu Cys Tyr Pro Thr Ty - #r Leu Tyr Gln Lys Ile           710                 7 - #15                 7 - #20                 7 -       #25                                                                           - GAT GAG TCA AAA TTA AAA GCC TAT ACT CGC TA - #T GAA TTA AGA GGG TAT         2321                                                                          Asp Glu Ser Lys Leu Lys Ala Tyr Thr Arg Ty - #r Glu Leu Arg Gly Tyr           #               740                                                           - ATT GAA GAT AGT CAA GAT TTA GAA GTC TAT TT - #G ATT CGT TAC AAT GCG         2369                                                                          Ile Glu Asp Ser Gln Asp Leu Glu Val Tyr Le - #u Ile Arg Tyr Asn Ala           #           755                                                               - AAA CAT GAA ACA GTA AAT GTT CCC GGT ACA GG - #G TCC TTA TGG CCG CTT         2417                                                                          Lys His Glu Thr Val Asn Val Pro Gly Thr Gl - #y Ser Leu Trp Pro Leu           #       770                                                                   - TCA GTC GAA AGC CCA ATC GGA AGG TGC GGA GA - #A CCG AAT CGA TGT GTG         2465                                                                          Ser Val Glu Ser Pro Ile Gly Arg Cys Gly Gl - #u Pro Asn Arg Cys Val           #   785                                                                       - CCA CAT ATT GAA TGG AAT CCT GAT TTA GAT TG - #T TCG TGT AGG GAT GGG         2513                                                                          Pro His Ile Glu Trp Asn Pro Asp Leu Asp Cy - #s Ser Cys Arg Asp Gly           790                 7 - #95                 8 - #00                 8 -       #05                                                                           - GAG AAG TGT GCC CAT CAT TCG CAT CAT TTC TC - #T CTA GAT ATT GAT GTT         2561                                                                          Glu Lys Cys Ala His His Ser His His Phe Se - #r Leu Asp Ile Asp Val           #               820                                                           - GGA TGT ACA GAC CTA AAT GAG GAC CTA GGT GT - #A TGG GTG ATC TTT AAG         2609                                                                          Gly Cys Thr Asp Leu Asn Glu Asp Leu Gly Va - #l Trp Val Ile Phe Lys           #           835                                                               - ATT AAA ACG CAG GAT GGC CAT GCA AGA TTA GG - #A AAT CTA GAG TTT CTC         2657                                                                          Ile Lys Thr Gln Asp Gly His Ala Arg Leu Gl - #y Asn Leu Glu Phe Leu           #       850                                                                   - GAA GAG AAA CCA TTG TTA GGA GAA GCG TTA GC - #T CGT GTG AAA AGA GCG         2705                                                                          Glu Glu Lys Pro Leu Leu Gly Glu Ala Leu Al - #a Arg Val Lys Arg Ala           #   865                                                                       - GAG AAA AAA TGG AGA GAC AAA CGC GAA CAA TT - #G CAG TTT GAA ACG AAT         2753                                                                          Glu Lys Lys Trp Arg Asp Lys Arg Glu Gln Le - #u Gln Phe Glu Thr Asn           870                 8 - #75                 8 - #80                 8 -       #85                                                                           - ATC GTT TAC AAA GAG GCA AAA GAA TCT GTA GA - #T GCT TTA TTC GTA GAT         2801                                                                          Ile Val Tyr Lys Glu Ala Lys Glu Ser Val As - #p Ala Leu Phe Val Asp           #               900                                                           - TCT CAC TAT AAT AGA TTA CAA GCG GAT ACG AA - #C ATT ACG ATG ATT CAT         2849                                                                          Ser His Tyr Asn Arg Leu Gln Ala Asp Thr As - #n Ile Thr Met Ile His           #           915                                                               - GCG GCA GAT AAA CGC GTT CAT CGA ATC CGA GA - #G GCT TAT CTT CCG GAA         2897                                                                          Ala Ala Asp Lys Arg Val His Arg Ile Arg Gl - #u Ala Tyr Leu Pro Glu           #       930                                                                   - TTA TCC GTT ATC CCA GGT GTA AAT GCG GAC AT - #T TTT GAA GAA TTA GAA         2945                                                                          Leu Ser Val Ile Pro Gly Val Asn Ala Asp Il - #e Phe Glu Glu Leu Glu           #   945                                                                       - GGT CTT ATT TTC ACT GCA TTC TCC CTA TAT GA - #T GCG AGA AAT ATC ATT         2993                                                                          Gly Leu Ile Phe Thr Ala Phe Ser Leu Tyr As - #p Ala Arg Asn Ile Ile           950                 9 - #55                 9 - #60                 9 -       #65                                                                           - AAA AAC GGT GAT TTC AAT AAT GGT TTA TCG TG - #T TGG AAC GTG AAA GGG         3041                                                                          Lys Asn Gly Asp Phe Asn Asn Gly Leu Ser Cy - #s Trp Asn Val Lys Gly           #               980                                                           - CAT GTA GAT ATA CAA CAG AAT GAT CAT CGT TC - #T GTC CTC GTT GTC CCG         3089                                                                          His Val Asp Ile Gln Gln Asn Asp His Arg Se - #r Val Leu Val Val Pro           #           995                                                               - GAA TGG GAA TCA GAG GTA TCA CAA GAA GTC CG - #C GTA TGT CCA GGT CGT         3137                                                                          Glu Trp Glu Ser Glu Val Ser Gln Glu Val Ar - #g Val Cys Pro Gly Arg           #      10105                                                                  - GGC TAT ATT CTT CGT GTC ACA GCG TAC AAA GA - #G GGC TAC GGA GAA GGA         3185                                                                          Gly Tyr Ile Leu Arg Val Thr Ala Tyr Lys Gl - #u Gly Tyr Gly Glu Gly           #  10250                                                                      - TGC GTA ACG ATC CAT GAG ATC GAA GAC AAT AC - #A GAC GAA TTG AAG TTT         3233                                                                          Cys Val Thr Ile His Glu Ile Glu Asp Asn Th - #r Asp Glu Leu Lys Phe           #               10451035 - #                1040                              - AGT AAC TGC ATA GAA GAG GAA GTC TAT CCA AC - #G GAT ACA GGT AAT GAT         3281                                                                          Ser Asn Cys Ile Glu Glu Glu Val Tyr Pro Th - #r Asp Thr Gly Asn Asp           #              10605                                                          - TAT ACT GCA CAC CAA GGT ACA ACA GGA TGC GC - #A GAT GCA TGT AAT TCC         3329                                                                          Tyr Thr Ala His Gln Gly Thr Thr Gly Cys Al - #a Asp Ala Cys Asn Ser           #          10750                                                              - CGT AAT GTT GGA TAT GAG GAT GGA TAT GAA AT - #A AAT ACT ACA GCA TCT         3377                                                                          Arg Asn Val Gly Tyr Glu Asp Gly Tyr Glu Il - #e Asn Thr Thr Ala Ser           #      10905                                                                  - GTT AAT TAC AAA CCG ACT TAT GAA GAA GAA AT - #G TAT ACA GAT GTA CGA         3425                                                                          Val Asn Tyr Lys Pro Thr Tyr Glu Glu Glu Me - #t Tyr Thr Asp Val Arg           #  11050                                                                      - AGA GAT AAT CAT TGT GAA TAT GAC AGA GGA TA - #T GGG AAC CAT ACA CCG         3473                                                                          Arg Asp Asn His Cys Glu Tyr Asp Arg Gly Ty - #r Gly Asn His Thr Pro           #               11251115 - #                1120                              - TTA CCA GCT GGT TAT GTA ACA AAA GAA TTA GA - #G TAC TTC CCT GAA ACA         3521                                                                          Leu Pro Ala Gly Tyr Val Thr Lys Glu Leu Gl - #u Tyr Phe Pro Glu Thr           #              11405                                                          - GAT ACA GTA TGG ATA GAG ATT GGA GAA ACG GA - #A GGA ACA TTC ATC GTA         3569                                                                          Asp Thr Val Trp Ile Glu Ile Gly Glu Thr Gl - #u Gly Thr Phe Ile Val           #          11550                                                              - GAT AGT GTG GAA TTA CTC CTC ATG GAG GAA TA - #AGATTGTA CGAAATCGAC           3619                                                                          Asp Ser Val Glu Leu Leu Leu Met Glu Glu                                       #       1165                                                                  - TTTAAATGGC TCATTCTAAA CAAAAAGTAG TCGTCTAATC TCTGTAACAA AT - #AGAAAAGT       3679                                                                          #      3713        AAGA AAAAGGACAT TACT                                       - (2) INFORMATION FOR SEQ ID NO:2:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 1167 amino                                                        (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                 - Met Glu Ile Asn Asn Gln Lys Gln Cys Ile Pr - #o Tyr Asn Cys Leu Ser         #                 15                                                          - Asn Pro Glu Glu Val Leu Leu Asp Gly Glu Ar - #g Ile Leu Pro Asp Ile         #             30                                                              - Asp Pro Leu Glu Val Ser Leu Ser Leu Leu Gl - #n Phe Leu Leu Asn Asn         #         45                                                                  - Phe Val Pro Gly Gly Gly Phe Ile Ser Gly Le - #u Val Asp Lys Ile Trp         #     60                                                                      - Gly Ala Leu Arg Pro Ser Glu Trp Asp Leu Ph - #e Leu Ala Gln Ile Glu         # 80                                                                          - Arg Leu Ile Asp Gln Arg Ile Glu Ala Thr Va - #l Arg Ala Lys Ala Ile         #                 95                                                          - Thr Glu Leu Glu Gly Leu Gly Arg Asn Tyr Gl - #n Ile Tyr Ala Glu Ala         #           110                                                               - Phe Lys Glu Trp Glu Ser Asp Pro Asp Asn Gl - #u Ala Ala Lys Ser Arg         #       125                                                                   - Val Ile Asp Arg Phe Arg Ile Leu Asp Gly Le - #u Ile Glu Ala Asn Ile         #   140                                                                       - Pro Ser Phe Arg Ile Ile Gly Phe Glu Val Pr - #o Leu Leu Ser Val Tyr         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Val Gln Ala Ala Asn Leu His Leu Ala Leu Le - #u Arg Asp Ser Val Ile         #               175                                                           - Phe Gly Glu Arg Trp Gly Leu Thr Thr Lys As - #n Val Asn Asp Ile Tyr         #           190                                                               - Asn Arg Gln Ile Arg Glu Ile His Glu Tyr Se - #r Asn His Cys Val Asp         #       205                                                                   - Thr Tyr Asn Thr Glu Leu Glu Arg Leu Gly Ph - #e Arg Ser Ile Ala Gln         #   220                                                                       - Trp Arg Ile Tyr Asn Gln Phe Arg Arg Glu Le - #u Thr Leu Thr Val Leu         225                 2 - #30                 2 - #35                 2 -       #40                                                                           - Asp Ile Val Ala Leu Phe Pro Asn Tyr Asp Se - #r Arg Leu Tyr Pro Ile         #               255                                                           - Gln Thr Phe Ser Gln Leu Thr Arg Glu Ile Va - #l Thr Ser Pro Val Ser         #           270                                                               - Glu Phe Tyr Tyr Gly Val Ile Asn Ser Gly As - #n Ile Ile Gly Thr Leu         #       285                                                                   - Thr Glu Gln Gln Ile Arg Arg Pro His Leu Me - #t Asp Phe Phe Asn Ser         #   300                                                                       - Met Ile Met Tyr Thr Ser Asp Asn Arg Arg Gl - #u His Tyr Trp Ser Gly         305                 3 - #10                 3 - #15                 3 -       #20                                                                           - Leu Glu Met Thr Ala Tyr Phe Thr Gly Phe Al - #a Gly Ala Gln Val Ser         #               335                                                           - Phe Pro Leu Val Gly Thr Arg Gly Glu Ser Al - #a Pro Pro Leu Thr Val         #           350                                                               - Arg Ser Val Asn Asp Gly Ile Tyr Arg Ile Le - #u Ser Ala Pro Phe Tyr         #       365                                                                   - Ser Ala Pro Phe Leu Gly Thr Ile Val Leu Gl - #y Ser Arg Gly Glu Lys         #   380                                                                       - Phe Asp Phe Ala Leu Asn Asn Ile Ser Pro Pr - #o Pro Ser Thr Ile Tyr         385                 3 - #90                 3 - #95                 4 -       #00                                                                           - Arg His Pro Gly Thr Val Asp Ser Leu Val Se - #r Ile Pro Pro Gln Asp         #               415                                                           - Asn Ser Val Pro Pro His Arg Gly Ser Ser Hi - #s Arg Leu Ser His Val         #           430                                                               - Thr Met Arg Ala Ser Ser Pro Ile Phe His Tr - #p Thr His Arg Ser Ala         #       445                                                                   - Thr Thr Thr Asn Thr Ile Asn Pro Asn Ala Il - #e Ile Gln Ile Pro Leu         #   460                                                                       - Val Lys Ala Phe Asn Leu His Ser Gly Ala Th - #r Val Val Arg Gly Pro         465                 4 - #70                 4 - #75                 4 -       #80                                                                           - Gly Phe Thr Gly Gly Asp Ile Leu Arg Arg Th - #r Asn Thr Gly Thr Phe         #               495                                                           - Ala Asp Met Arg Val Asn Ile Thr Gly Pro Le - #u Ser Gln Arg Tyr Arg         #           510                                                               - Val Arg Ile Arg Tyr Ala Ser Thr Thr Asp Le - #u Gln Phe Phe Thr Arg         #       525                                                                   - Ile Asn Gly Thr Ser Val Asn Gln Gly Asn Ph - #e Gln Arg Thr Met Asn         #   540                                                                       - Arg Gly Asp Asn Leu Glu Ser Gly Asn Phe Ar - #g Thr Ala Gly Phe Ser         545                 5 - #50                 5 - #55                 5 -       #60                                                                           - Thr Pro Phe Ser Phe Ser Asn Ala Gln Ser Th - #r Phe Thr Leu Gly Thr         #               575                                                           - Gln Ala Phe Ser Asn Gln Glu Val Tyr Ile As - #p Arg Ile Glu Phe Val         #           590                                                               - Pro Ala Glu Val Thr Phe Glu Ala Glu Ser As - #p Leu Glu Arg Ala Gln         #       605                                                                   - Lys Ala Val Asn Ala Leu Phe Thr Ser Thr As - #n Gln Leu Gly Leu Lys         #   620                                                                       - Thr Asp Val Thr Asp Tyr Gln Ile Asp Gln Va - #l Ser Asn Leu Val Glu         625                 6 - #30                 6 - #35                 6 -       #40                                                                           - Cys Leu Ser Asp Glu Phe Cys Leu Asp Glu Ly - #s Arg Glu Leu Ser Glu         #               655                                                           - Lys Val Lys His Ala Lys Arg Leu Ser Asp Ly - #s Arg Asn Leu Leu Gln         #           670                                                               - Asp Pro Asn Phe Thr Ser Ile Asn Arg Gln Le - #u Asp Arg Gly Trp Arg         #       685                                                                   - Gly Ser Thr Asp Ile Thr Ile Gln Gly Gly As - #n Asp Val Phe Lys Glu         #   700                                                                       - Asn Tyr Val Thr Leu Pro Gly Thr Phe Asp Gl - #u Cys Tyr Pro Thr Tyr         705                 7 - #10                 7 - #15                 7 -       #20                                                                           - Leu Tyr Gln Lys Ile Asp Glu Ser Lys Leu Ly - #s Ala Tyr Thr Arg Tyr         #               735                                                           - Glu Leu Arg Gly Tyr Ile Glu Asp Ser Gln As - #p Leu Glu Val Tyr Leu         #           750                                                               - Ile Arg Tyr Asn Ala Lys His Glu Thr Val As - #n Val Pro Gly Thr Gly         #       765                                                                   - Ser Leu Trp Pro Leu Ser Val Glu Ser Pro Il - #e Gly Arg Cys Gly Glu         #   780                                                                       - Pro Asn Arg Cys Val Pro His Ile Glu Trp As - #n Pro Asp Leu Asp Cys         785                 7 - #90                 7 - #95                 8 -       #00                                                                           - Ser Cys Arg Asp Gly Glu Lys Cys Ala His Hi - #s Ser His His Phe Ser         #               815                                                           - Leu Asp Ile Asp Val Gly Cys Thr Asp Leu As - #n Glu Asp Leu Gly Val         #           830                                                               - Trp Val Ile Phe Lys Ile Lys Thr Gln Asp Gl - #y His Ala Arg Leu Gly         #       845                                                                   - Asn Leu Glu Phe Leu Glu Glu Lys Pro Leu Le - #u Gly Glu Ala Leu Ala         #   860                                                                       - Arg Val Lys Arg Ala Glu Lys Lys Trp Arg As - #p Lys Arg Glu Gln Leu         865                 8 - #70                 8 - #75                 8 -       #80                                                                           - Gln Phe Glu Thr Asn Ile Val Tyr Lys Glu Al - #a Lys Glu Ser Val Asp         #               895                                                           - Ala Leu Phe Val Asp Ser His Tyr Asn Arg Le - #u Gln Ala Asp Thr Asn         #           910                                                               - Ile Thr Met Ile His Ala Ala Asp Lys Arg Va - #l His Arg Ile Arg Glu         #       925                                                                   - Ala Tyr Leu Pro Glu Leu Ser Val Ile Pro Gl - #y Val Asn Ala Asp Ile         #   940                                                                       - Phe Glu Glu Leu Glu Gly Leu Ile Phe Thr Al - #a Phe Ser Leu Tyr Asp         945                 9 - #50                 9 - #55                 9 -       #60                                                                           - Ala Arg Asn Ile Ile Lys Asn Gly Asp Phe As - #n Asn Gly Leu Ser Cys         #               975                                                           - Trp Asn Val Lys Gly His Val Asp Ile Gln Gl - #n Asn Asp His Arg Ser         #           990                                                               - Val Leu Val Val Pro Glu Trp Glu Ser Glu Va - #l Ser Gln Glu Val Arg         #      10050                                                                  - Val Cys Pro Gly Arg Gly Tyr Ile Leu Arg Va - #l Thr Ala Tyr Lys Glu         #  10205                                                                      - Gly Tyr Gly Glu Gly Cys Val Thr Ile His Gl - #u Ile Glu Asp Asn Thr         #               10401030 - #                1035                              - Asp Glu Leu Lys Phe Ser Asn Cys Ile Glu Gl - #u Glu Val Tyr Pro Thr         #              10550                                                          - Asp Thr Gly Asn Asp Tyr Thr Ala His Gln Gl - #y Thr Thr Gly Cys Ala         #          10705                                                              - Asp Ala Cys Asn Ser Arg Asn Val Gly Tyr Gl - #u Asp Gly Tyr Glu Ile         #      10850                                                                  - Asn Thr Thr Ala Ser Val Asn Tyr Lys Pro Th - #r Tyr Glu Glu Glu Met         #  11005                                                                      - Tyr Thr Asp Val Arg Arg Asp Asn His Cys Gl - #u Tyr Asp Arg Gly Tyr         #               11201110 - #                1115                              - Gly Asn His Thr Pro Leu Pro Ala Gly Tyr Va - #l Thr Lys Glu Leu Glu         #              11350                                                          - Tyr Phe Pro Glu Thr Asp Thr Val Trp Ile Gl - #u Ile Gly Glu Thr Glu         #          11505                                                              - Gly Thr Phe Ile Val Asp Ser Val Glu Leu Le - #u Leu Met Glu Glu             #      11650                                                                  - (2) INFORMATION FOR SEQ ID NO:3:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 3934 base                                                         (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: circular                                              -     (ii) MOLECULE TYPE: DNA (genomic)                                       -     (ix) FEATURE:                                                                     (A) NAME/KEY: CDS                                                             (B) LOCATION: 67..3756                                              -     (ix) FEATURE:                                                                     (A) NAME/KEY: misc.sub.-- - #feature                                          (B) LOCATION: 2253..2272                                            -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                 - AAACTATTCA ATGGAGAAAA ATTGAATAGT TGTAATGTAA GCACACCGAA AA - #AAGGAGGA         60                                                                          #GAA ATT ATA AAT GCT       108AA AAT GAG AAT                                         Leu Thr Ser Asn Arg Lys As - #n Glu Asn Glu Ile Ile Asn Ala            #      10                                                                     - TTA TCG ATT CCA ACG GTA TCG AAT CCT TCC AC - #G CAA ATG AAT CTA TCA          156                                                                          Leu Ser Ile Pro Thr Val Ser Asn Pro Ser Th - #r Gln Met Asn Leu Ser           # 30                                                                          - CCA GAT GCT CGT ATT GAA GAT AGC TTG TGT GT - #A GCC GAG GTG AAC AAT          204                                                                          Pro Asp Ala Arg Ile Glu Asp Ser Leu Cys Va - #l Ala Glu Val Asn Asn           #                 45                                                          - ATT GAT CCA TTT GTT AGC GCA TCA ACA GTC CA - #A ACG GGT ATA AAC ATA          252                                                                          Ile Asp Pro Phe Val Ser Ala Ser Thr Val Gl - #n Thr Gly Ile Asn Ile           #             60                                                              - GCT GGT AGA ATA TTG GGC GTA TTA GGT GTG CC - #G TTT GCT GGA CAA CTA          300                                                                          Ala Gly Arg Ile Leu Gly Val Leu Gly Val Pr - #o Phe Ala Gly Gln Leu           #         75                                                                  - GCT AGT TTT TAT AGT TTT CTT GTT GGG GAA TT - #A TGG CCT AGT GGC AGA          348                                                                          Ala Ser Phe Tyr Ser Phe Leu Val Gly Glu Le - #u Trp Pro Ser Gly Arg           #     90                                                                      - GAT CCA TGG GAA ATT TTC CTG GAA CAT GTA GA - #A CAA CTT ATA AGA CAA          396                                                                          Asp Pro Trp Glu Ile Phe Leu Glu His Val Gl - #u Gln Leu Ile Arg Gln           #110                                                                          - CAA GTA ACA GAA AAT ACT AGG AAT ACG GCT AT - #T GCT CGA TTA GAA GGT          444                                                                          Gln Val Thr Glu Asn Thr Arg Asn Thr Ala Il - #e Ala Arg Leu Glu Gly           #               125                                                           - CTA GGA AGA GGC TAT AGA TCT TAC CAG CAG GC - #T CTT GAA ACT TGG TTA          492                                                                          Leu Gly Arg Gly Tyr Arg Ser Tyr Gln Gln Al - #a Leu Glu Thr Trp Leu           #           140                                                               - GAT AAC CGA AAT GAT GCA AGA TCA AGA AGC AT - #T ATT CTT GAG CGC TAT          540                                                                          Asp Asn Arg Asn Asp Ala Arg Ser Arg Ser Il - #e Ile Leu Glu Arg Tyr           #       155                                                                   - GTT GCT TTA GAA CTT GAC ATT ACT ACT GCT AT - #A CCG CTT TTC AGA ATA          588                                                                          Val Ala Leu Glu Leu Asp Ile Thr Thr Ala Il - #e Pro Leu Phe Arg Ile           #   170                                                                       - CGA AAT GAA GAA GTT CCA TTA TTA ATG GTA TA - #T GCT CAA GCT GCA AAT          636                                                                          Arg Asn Glu Glu Val Pro Leu Leu Met Val Ty - #r Ala Gln Ala Ala Asn           175                 1 - #80                 1 - #85                 1 -       #90                                                                           - TTA CAC CTA TTA TTA TTG AGA GAC GCA TCC CT - #T TTT GGT AGT GAA TGG          684                                                                          Leu His Leu Leu Leu Leu Arg Asp Ala Ser Le - #u Phe Gly Ser Glu Trp           #               205                                                           - GGG ATG GCA TCT TCC GAT GTT AAC CAA TAT TA - #C CAA GAA CAA ATC AGA          732                                                                          Gly Met Ala Ser Ser Asp Val Asn Gln Tyr Ty - #r Gln Glu Gln Ile Arg           #           220                                                               - TAT ACA GAG GAA TAT TCT AAC CAT TGC GTA CA - #A TGG TAT AAT ACA GGG          780                                                                          Tyr Thr Glu Glu Tyr Ser Asn His Cys Val Gl - #n Trp Tyr Asn Thr Gly           #       235                                                                   - CTA AAT AAC TTA AGA GGG ACA AAT GCT GAA AG - #T TGG TTG CGG TAT AAT          828                                                                          Leu Asn Asn Leu Arg Gly Thr Asn Ala Glu Se - #r Trp Leu Arg Tyr Asn           #   250                                                                       - CAA TTC CGT AGA GAC CTA ACG TTA GGG GTA TT - #A GAT TTA GTA GCC CTA          876                                                                          Gln Phe Arg Arg Asp Leu Thr Leu Gly Val Le - #u Asp Leu Val Ala Leu           255                 2 - #60                 2 - #65                 2 -       #70                                                                           - TTC CCA AGC TAT GAT ACT CGC ACT TAT CCA AT - #C AAT ACG AGT GCT CAG          924                                                                          Phe Pro Ser Tyr Asp Thr Arg Thr Tyr Pro Il - #e Asn Thr Ser Ala Gln           #               285                                                           - TTA ACA AGA GAA ATT TAT ACA GAT CCA ATT GG - #G AGA ACA AAT GCA CCT          972                                                                          Leu Thr Arg Glu Ile Tyr Thr Asp Pro Ile Gl - #y Arg Thr Asn Ala Pro           #           300                                                               - TCA GGA TTT GCA AGT ACG AAT TGG TTT AAT AA - #T AAT GCA CCA TCG TTT         1020                                                                          Ser Gly Phe Ala Ser Thr Asn Trp Phe Asn As - #n Asn Ala Pro Ser Phe           #       315                                                                   - TCT GCC ATA GAG GCT GCC ATT TTC AGG CCT CC - #G CAT CTA CTT GAT TTT         1068                                                                          Ser Ala Ile Glu Ala Ala Ile Phe Arg Pro Pr - #o His Leu Leu Asp Phe           #   330                                                                       - CCA GAA CAA CTT ACA ATT TAC AGT GCA TCA AG - #C CGT TGG AGT AGC ACT         1116                                                                          Pro Glu Gln Leu Thr Ile Tyr Ser Ala Ser Se - #r Arg Trp Ser Ser Thr           335                 3 - #40                 3 - #45                 3 -       #50                                                                           - CAA CAT ATG AAT TAT TGG GTG GGA CAT AGG CT - #T AAC TTC CGC CCA ATA         1164                                                                          Gln His Met Asn Tyr Trp Val Gly His Arg Le - #u Asn Phe Arg Pro Ile           #               365                                                           - GGA GGG ACA TTA AAT ACC TCA ACA CAA GGA CT - #T ACT AAT AAT ACT TCA         1212                                                                          Gly Gly Thr Leu Asn Thr Ser Thr Gln Gly Le - #u Thr Asn Asn Thr Ser           #           380                                                               - ATT AAT CCT GTA ACA TTA CAG TTT ACG TCT CG - #A GAC GTT TAT AGA ACA         1260                                                                          Ile Asn Pro Val Thr Leu Gln Phe Thr Ser Ar - #g Asp Val Tyr Arg Thr           #       395                                                                   - GAA TCA AAT GCA GGG ACA AAT ATA CTA TTT AC - #T ACT CCT GTG AAT GGA         1308                                                                          Glu Ser Asn Ala Gly Thr Asn Ile Leu Phe Th - #r Thr Pro Val Asn Gly           #   410                                                                       - GTA CCT TGG GCT AGA TTT AAT TTT ATA AAC CC - #T CAG AAT ATT TAT GAA         1356                                                                          Val Pro Trp Ala Arg Phe Asn Phe Ile Asn Pr - #o Gln Asn Ile Tyr Glu           415                 4 - #20                 4 - #25                 4 -       #30                                                                           - AGA GGC GCC ACT ACC TAC AGT CAA CCG TAT CA - #G GGA GTT GGG ATT CAA         1404                                                                          Arg Gly Ala Thr Thr Tyr Ser Gln Pro Tyr Gl - #n Gly Val Gly Ile Gln           #               445                                                           - TTA TTT GAT TCA GAA ACT GAA TTA CCA CCA GA - #A ACA ACA GAA CGA CCA         1452                                                                          Leu Phe Asp Ser Glu Thr Glu Leu Pro Pro Gl - #u Thr Thr Glu Arg Pro           #           460                                                               - AAT TAT GAA TCA TAT AGT CAT AGA TTA TCT CA - #T ATA GGA CTA ATC ATA         1500                                                                          Asn Tyr Glu Ser Tyr Ser His Arg Leu Ser Hi - #s Ile Gly Leu Ile Ile           #       475                                                                   - GGA AAC ACT TTG AGA GCA CCA GTC TAT TCT TG - #G ACG CAT CGT AGT GCA         1548                                                                          Gly Asn Thr Leu Arg Ala Pro Val Tyr Ser Tr - #p Thr His Arg Ser Ala           #   490                                                                       - GAT CGT ACG AAT ACG ATT GGA CCA AAT AGA AT - #T ACA CAA ATA CCA TTG         1596                                                                          Asp Arg Thr Asn Thr Ile Gly Pro Asn Arg Il - #e Thr Gln Ile Pro Leu           495                 5 - #00                 5 - #05                 5 -       #10                                                                           - GTA AAA GCA CTG AAT CTT CAT TCA GGT GTT AC - #T GTT GTT GGA GGG CCA         1644                                                                          Val Lys Ala Leu Asn Leu His Ser Gly Val Th - #r Val Val Gly Gly Pro           #               525                                                           - GGA TTT ACA GGT GGG GAT ATC CTT CGT AGA AC - #A AAT ACG GGT ACA TTT         1692                                                                          Gly Phe Thr Gly Gly Asp Ile Leu Arg Arg Th - #r Asn Thr Gly Thr Phe           #           540                                                               - GGA GAT ATA CGA TTA AAT ATT AAT GTG CCA TT - #A TCC CAA AGA TAT CGC         1740                                                                          Gly Asp Ile Arg Leu Asn Ile Asn Val Pro Le - #u Ser Gln Arg Tyr Arg           #       555                                                                   - GTA AGG ATT CGT TAT GCT TCT ACT ACA GAT TT - #A CAA TTT TTC ACG AGA         1788                                                                          Val Arg Ile Arg Tyr Ala Ser Thr Thr Asp Le - #u Gln Phe Phe Thr Arg           #   570                                                                       - ATT AAT GGA ACC ACT GTT AAT ATT GGT AAT TT - #C TCA AGA ACT ATG AAT         1836                                                                          Ile Asn Gly Thr Thr Val Asn Ile Gly Asn Ph - #e Ser Arg Thr Met Asn           575                 5 - #80                 5 - #85                 5 -       #90                                                                           - AGG GGG GAT AAT TTA GAA TAT AGA AGT TTT AG - #A ACT GCA GGA TTT AGT         1884                                                                          Arg Gly Asp Asn Leu Glu Tyr Arg Ser Phe Ar - #g Thr Ala Gly Phe Ser           #               605                                                           - ACT CCT TTT AAT TTT TTA AAT GCC CAA AGC AC - #A TTC ACA TTG GGT GCT         1932                                                                          Thr Pro Phe Asn Phe Leu Asn Ala Gln Ser Th - #r Phe Thr Leu Gly Ala           #           620                                                               - CAG AGT TTT TCA AAT CAG GAA GTT TAT ATA GA - #T AGA GTC GAA TTT GTT         1980                                                                          Gln Ser Phe Ser Asn Gln Glu Val Tyr Ile As - #p Arg Val Glu Phe Val           #       635                                                                   - CCA GCA GAG GTA ACA TTT GAG GCA GAA TAT GA - #T TTA GAA AGA GCA CAA         2028                                                                          Pro Ala Glu Val Thr Phe Glu Ala Glu Tyr As - #p Leu Glu Arg Ala Gln           #   650                                                                       - AAG GCG GTG AAT GCT CTG TTT ACT TCT ACA AA - #T CCA AGA AGA TTG AAA         2076                                                                          Lys Ala Val Asn Ala Leu Phe Thr Ser Thr As - #n Pro Arg Arg Leu Lys           655                 6 - #60                 6 - #65                 6 -       #70                                                                           - ACA GAT GTG ACA GAT TAT CAT ATT GAC CAA GT - #G TCC AAT ATG GTG GCA         2124                                                                          Thr Asp Val Thr Asp Tyr His Ile Asp Gln Va - #l Ser Asn Met Val Ala           #               685                                                           - TGT TTA TCA GAT GAA TTT TGC TTG GAT GAG AA - #G CGA GAA TTA TTT GAG         2172                                                                          Cys Leu Ser Asp Glu Phe Cys Leu Asp Glu Ly - #s Arg Glu Leu Phe Glu           #           700                                                               - AAA GTG AAA TAT GCG AAG CGA CTC AGT GAT GA - #A AGA AAC TTA CTC CAA         2220                                                                          Lys Val Lys Tyr Ala Lys Arg Leu Ser Asp Gl - #u Arg Asn Leu Leu Gln           #       715                                                                   - GAT CCA AAC TTC ACA TTC ATC AGT GGG CAA TT - #A AGT TTC GCA TCC ATC         2268                                                                          Asp Pro Asn Phe Thr Phe Ile Ser Gly Gln Le - #u Ser Phe Ala Ser Ile           #   730                                                                       - GAT GGA CAA TCA AAC TTC CCC TCT ATT AAT GA - #G CTA TCT GAA CAT GGA         2316                                                                          Asp Gly Gln Ser Asn Phe Pro Ser Ile Asn Gl - #u Leu Ser Glu His Gly           735                 7 - #40                 7 - #45                 7 -       #50                                                                           - TGG TGG GGA AGT GCG AAT GTT ACC ATT CAG GA - #A GGG AAT GAC GTA TTT         2364                                                                          Trp Trp Gly Ser Ala Asn Val Thr Ile Gln Gl - #u Gly Asn Asp Val Phe           #               765                                                           - AAA GAG AAT TAC GTC ACA CTA CCG GGT ACT TT - #T AAT GAG TGT TAT CCA         2412                                                                          Lys Glu Asn Tyr Val Thr Leu Pro Gly Thr Ph - #e Asn Glu Cys Tyr Pro           #           780                                                               - AAT TAT TTA TAT CAA AAA ATA GGA GAG TCA GA - #A TTA AAA GCT TAT ACG         2460                                                                          Asn Tyr Leu Tyr Gln Lys Ile Gly Glu Ser Gl - #u Leu Lys Ala Tyr Thr           #       795                                                                   - CGC TAT CAA TTA AGA GGG TAT ATT GAA GAT AG - #T CAA GAT CTA GAG ATT         2508                                                                          Arg Tyr Gln Leu Arg Gly Tyr Ile Glu Asp Se - #r Gln Asp Leu Glu Ile           #   810                                                                       - TAT TTA ATT CGT TAC AAT GCA AAG CAT GAA AC - #A TTG GAT GTT CCA GGT         2556                                                                          Tyr Leu Ile Arg Tyr Asn Ala Lys His Glu Th - #r Leu Asp Val Pro Gly           815                 8 - #20                 8 - #25                 8 -       #30                                                                           - ACC GAT TCC CTA TGG CCG CTT TCA GTT GAA AG - #C CCA ATC GGA AGG TGC         2604                                                                          Thr Asp Ser Leu Trp Pro Leu Ser Val Glu Se - #r Pro Ile Gly Arg Cys           #               845                                                           - GGA GAA CCA AAT CGA TGC GCA CCA CAT TTT GA - #A TGG AAT CCT GAT CTA         2652                                                                          Gly Glu Pro Asn Arg Cys Ala Pro His Phe Gl - #u Trp Asn Pro Asp Leu           #           860                                                               - GAT TGT TCC TGC AGA GAT GGA GAA AGA TGT GC - #G CAT CAT TCC CAT CAT         2700                                                                          Asp Cys Ser Cys Arg Asp Gly Glu Arg Cys Al - #a His His Ser His His           #       875                                                                   - TTC ACT TTG GAT ATT GAT GTT GGG TGC ACA GA - #C TTG CAT GAG AAC CTA         2748                                                                          Phe Thr Leu Asp Ile Asp Val Gly Cys Thr As - #p Leu His Glu Asn Leu           #   890                                                                       - GGC GTG TGG GTG GTA TTC AAG ATT AAG ACG CA - #G GAA GGT TAT GCA AGA         2796                                                                          Gly Val Trp Val Val Phe Lys Ile Lys Thr Gl - #n Glu Gly Tyr Ala Arg           895                 9 - #00                 9 - #05                 9 -       #10                                                                           - TTA GGA AAT CTG GAA TTT ATC GAA GAG AAA CC - #A TTA ATT GGA GAA GCA         2844                                                                          Leu Gly Asn Leu Glu Phe Ile Glu Glu Lys Pr - #o Leu Ile Gly Glu Ala           #               925                                                           - CTG TCT CGT GTG AAG AGA GCG GAA AAA AAA TG - #G AGA GAC AAA CGG GAA         2892                                                                          Leu Ser Arg Val Lys Arg Ala Glu Lys Lys Tr - #p Arg Asp Lys Arg Glu           #           940                                                               - AAA CTA CAA TTG GAA ACA AAA CGA GTA TAT AC - #A GAG GCA AAA GAA GCT         2940                                                                          Lys Leu Gln Leu Glu Thr Lys Arg Val Tyr Th - #r Glu Ala Lys Glu Ala           #       955                                                                   - GTG GAT GCT TTA TTC GTA GAT TCT CAA TAT GA - #T CAA TTA CAA GCG GAT         2988                                                                          Val Asp Ala Leu Phe Val Asp Ser Gln Tyr As - #p Gln Leu Gln Ala Asp           #   970                                                                       - ACA AAC ATT GGC ATG ATT CAT GCG GCA GAT AA - #A CTT GTT CAT CGA ATT         3036                                                                          Thr Asn Ile Gly Met Ile His Ala Ala Asp Ly - #s Leu Val His Arg Ile           975                 9 - #80                 9 - #85                 9 -       #90                                                                           - CGA GAG GCG TAT CTT TCA GAA TTA CCT GTT AT - #C CCA GGT GTA AAT GCG         3084                                                                          Arg Glu Ala Tyr Leu Ser Glu Leu Pro Val Il - #e Pro Gly Val Asn Ala           #              10050                                                          - GAA ATT TTT GAA GAA TTA GAA GGT CAC ATT AT - #C ACT GCA ATG TCC TTA         3132                                                                          Glu Ile Phe Glu Glu Leu Glu Gly His Ile Il - #e Thr Ala Met Ser Leu           #          10205                                                              - TAC GAT GCG AGA AAT GTC GTT AAA AAT GGT GA - #T TTT AAT AAT GGA TTA         3180                                                                          Tyr Asp Ala Arg Asn Val Val Lys Asn Gly As - #p Phe Asn Asn Gly Leu           #      10350                                                                  - ACA TGT TGG AAT GTA AAA GGG CAT GTA GAT GT - #A CAA CAG AGC CAT CAT         3228                                                                          Thr Cys Trp Asn Val Lys Gly His Val Asp Va - #l Gln Gln Ser His His           #  10505                                                                      - CGT TCT GAC CTT GTT ATC CCA GAA TGG GAA GC - #A GAA GTG TCA CAA GCA         3276                                                                          Arg Ser Asp Leu Val Ile Pro Glu Trp Glu Al - #a Glu Val Ser Gln Ala           #               10701060 - #                1065                              - GTT CGC GTC TGT CCG GGG CGT GGC TAT ATC CT - #T CGT GTC ACA GCG TAC         3324                                                                          Val Arg Val Cys Pro Gly Arg Gly Tyr Ile Le - #u Arg Val Thr Ala Tyr           #              10850                                                          - AAA GAG GGA TAT GGA GAG GGC TGC GTA ACG AT - #C CAT GAA ATC GAG AAC         3372                                                                          Lys Glu Gly Tyr Gly Glu Gly Cys Val Thr Il - #e His Glu Ile Glu Asn           #          11005                                                              - AAT ACA GAC GAA CTA AAA TTT AAA AAC TGT GA - #A GAA GAG GAA GTG TAT         3420                                                                          Asn Thr Asp Glu Leu Lys Phe Lys Asn Cys Gl - #u Glu Glu Glu Val Tyr           #      11150                                                                  - CCA ACG GAT ACA GGA ACG TGT AAT GAT TAT AC - #T GCA CAC CAA GGT ACA         3468                                                                          Pro Thr Asp Thr Gly Thr Cys Asn Asp Tyr Th - #r Ala His Gln Gly Thr           #  11305                                                                      - GCA GCA TGT AAT TCC CGT AAT GCT GGA TAT GA - #G GAT GCA TAT GAA GTT         3516                                                                          Ala Ala Cys Asn Ser Arg Asn Ala Gly Tyr Gl - #u Asp Ala Tyr Glu Val           #               11501140 - #                1145                              - GAT ACT ACA GCA TCT GTT AAT TAC AAA CCG AC - #T TAT GAA GAA GAA ACG         3564                                                                          Asp Thr Thr Ala Ser Val Asn Tyr Lys Pro Th - #r Tyr Glu Glu Glu Thr           #              11650                                                          - TAT ACA GAT GTA CGA AGA GAT AAT CAT TGT GA - #A TAT GAC AGA GGG TAT         3612                                                                          Tyr Thr Asp Val Arg Arg Asp Asn His Cys Gl - #u Tyr Asp Arg Gly Tyr           #          11805                                                              - GTG AAT TAT CCA CCA GTA CCA GCT GGT TAT GT - #G ACA AAA GAA TTA GAA         3660                                                                          Val Asn Tyr Pro Pro Val Pro Ala Gly Tyr Va - #l Thr Lys Glu Leu Glu           #      11950                                                                  - TAC TTC CCA GAA ACA GAT ACA GTA TGG ATT GA - #G ATT GGA GAA ACG GAA         3708                                                                          Tyr Phe Pro Glu Thr Asp Thr Val Trp Ile Gl - #u Ile Gly Glu Thr Glu           #  12105                                                                      - GGA AAG TTT ATT GTA GAT AGC GTG GAA CTA CT - #C CTC ATG GAA GAA             TAGGATCA3763                                                                  Gly Lys Phe Ile Val Asp Ser Val Glu Leu Le - #u Leu Met Glu Glu               #               123 1220 - #                1225                              - CAAGTATAGC AGTTTAATAA ATATTAATTA AAATAGTAGT CTAACTTCCG TT - #CCAATTAA       3823                                                                          - ATAAGTAAAT TACAGTTGTA AAAAGAAAAC GGACATCACT CTTCAGAGAG CG - #ATGTCCGT       3883                                                                          #           3934TGTGCTA ATGATAAATG TGCACGAAAT TATATTGTCA A                    - (2) INFORMATION FOR SEQ ID NO:4:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 1229 amino                                                        (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                 - Leu Thr Ser Asn Arg Lys Asn Glu Asn Glu Il - #e Ile Asn Ala Leu Ser         #                 15                                                          - Ile Pro Thr Val Ser Asn Pro Ser Thr Gln Me - #t Asn Leu Ser Pro Asp         #             30                                                              - Ala Arg Ile Glu Asp Ser Leu Cys Val Ala Gl - #u Val Asn Asn Ile Asp         #         45                                                                  - Pro Phe Val Ser Ala Ser Thr Val Gln Thr Gl - #y Ile Asn Ile Ala Gly         #     60                                                                      - Arg Ile Leu Gly Val Leu Gly Val Pro Phe Al - #a Gly Gln Leu Ala Ser         # 80                                                                          - Phe Tyr Ser Phe Leu Val Gly Glu Leu Trp Pr - #o Ser Gly Arg Asp Pro         #                 95                                                          - Trp Glu Ile Phe Leu Glu His Val Glu Gln Le - #u Ile Arg Gln Gln Val         #           110                                                               - Thr Glu Asn Thr Arg Asn Thr Ala Ile Ala Ar - #g Leu Glu Gly Leu Gly         #       125                                                                   - Arg Gly Tyr Arg Ser Tyr Gln Gln Ala Leu Gl - #u Thr Trp Leu Asp Asn         #   140                                                                       - Arg Asn Asp Ala Arg Ser Arg Ser Ile Ile Le - #u Glu Arg Tyr Val Ala         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Leu Glu Leu Asp Ile Thr Thr Ala Ile Pro Le - #u Phe Arg Ile Arg Asn         #               175                                                           - Glu Glu Val Pro Leu Leu Met Val Tyr Ala Gl - #n Ala Ala Asn Leu His         #           190                                                               - Leu Leu Leu Leu Arg Asp Ala Ser Leu Phe Gl - #y Ser Glu Trp Gly Met         #       205                                                                   - Ala Ser Ser Asp Val Asn Gln Tyr Tyr Gln Gl - #u Gln Ile Arg Tyr Thr         #   220                                                                       - Glu Glu Tyr Ser Asn His Cys Val Gln Trp Ty - #r Asn Thr Gly Leu Asn         225                 2 - #30                 2 - #35                 2 -       #40                                                                           - Asn Leu Arg Gly Thr Asn Ala Glu Ser Trp Le - #u Arg Tyr Asn Gln Phe         #               255                                                           - Arg Arg Asp Leu Thr Leu Gly Val Leu Asp Le - #u Val Ala Leu Phe Pro         #           270                                                               - Ser Tyr Asp Thr Arg Thr Tyr Pro Ile Asn Th - #r Ser Ala Gln Leu Thr         #       285                                                                   - Arg Glu Ile Tyr Thr Asp Pro Ile Gly Arg Th - #r Asn Ala Pro Ser Gly         #   300                                                                       - Phe Ala Ser Thr Asn Trp Phe Asn Asn Asn Al - #a Pro Ser Phe Ser Ala         305                 3 - #10                 3 - #15                 3 -       #20                                                                           - Ile Glu Ala Ala Ile Phe Arg Pro Pro His Le - #u Leu Asp Phe Pro Glu         #               335                                                           - Gln Leu Thr Ile Tyr Ser Ala Ser Ser Arg Tr - #p Ser Ser Thr Gln His         #           350                                                               - Met Asn Tyr Trp Val Gly His Arg Leu Asn Ph - #e Arg Pro Ile Gly Gly         #       365                                                                   - Thr Leu Asn Thr Ser Thr Gln Gly Leu Thr As - #n Asn Thr Ser Ile Asn         #   380                                                                       - Pro Val Thr Leu Gln Phe Thr Ser Arg Asp Va - #l Tyr Arg Thr Glu Ser         385                 3 - #90                 3 - #95                 4 -       #00                                                                           - Asn Ala Gly Thr Asn Ile Leu Phe Thr Thr Pr - #o Val Asn Gly Val Pro         #               415                                                           - Trp Ala Arg Phe Asn Phe Ile Asn Pro Gln As - #n Ile Tyr Glu Arg Gly         #           430                                                               - Ala Thr Thr Tyr Ser Gln Pro Tyr Gln Gly Va - #l Gly Ile Gln Leu Phe         #       445                                                                   - Asp Ser Glu Thr Glu Leu Pro Pro Glu Thr Th - #r Glu Arg Pro Asn Tyr         #   460                                                                       - Glu Ser Tyr Ser His Arg Leu Ser His Ile Gl - #y Leu Ile Ile Gly Asn         465                 4 - #70                 4 - #75                 4 -       #80                                                                           - Thr Leu Arg Ala Pro Val Tyr Ser Trp Thr Hi - #s Arg Ser Ala Asp Arg         #               495                                                           - Thr Asn Thr Ile Gly Pro Asn Arg Ile Thr Gl - #n Ile Pro Leu Val Lys         #           510                                                               - Ala Leu Asn Leu His Ser Gly Val Thr Val Va - #l Gly Gly Pro Gly Phe         #       525                                                                   - Thr Gly Gly Asp Ile Leu Arg Arg Thr Asn Th - #r Gly Thr Phe Gly Asp         #   540                                                                       - Ile Arg Leu Asn Ile Asn Val Pro Leu Ser Gl - #n Arg Tyr Arg Val Arg         545                 5 - #50                 5 - #55                 5 -       #60                                                                           - Ile Arg Tyr Ala Ser Thr Thr Asp Leu Gln Ph - #e Phe Thr Arg Ile Asn         #               575                                                           - Gly Thr Thr Val Asn Ile Gly Asn Phe Ser Ar - #g Thr Met Asn Arg Gly         #           590                                                               - Asp Asn Leu Glu Tyr Arg Ser Phe Arg Thr Al - #a Gly Phe Ser Thr Pro         #       605                                                                   - Phe Asn Phe Leu Asn Ala Gln Ser Thr Phe Th - #r Leu Gly Ala Gln Ser         #   620                                                                       - Phe Ser Asn Gln Glu Val Tyr Ile Asp Arg Va - #l Glu Phe Val Pro Ala         625                 6 - #30                 6 - #35                 6 -       #40                                                                           - Glu Val Thr Phe Glu Ala Glu Tyr Asp Leu Gl - #u Arg Ala Gln Lys Ala         #               655                                                           - Val Asn Ala Leu Phe Thr Ser Thr Asn Pro Ar - #g Arg Leu Lys Thr Asp         #           670                                                               - Val Thr Asp Tyr His Ile Asp Gln Val Ser As - #n Met Val Ala Cys Leu         #       685                                                                   - Ser Asp Glu Phe Cys Leu Asp Glu Lys Arg Gl - #u Leu Phe Glu Lys Val         #   700                                                                       - Lys Tyr Ala Lys Arg Leu Ser Asp Glu Arg As - #n Leu Leu Gln Asp Pro         705                 7 - #10                 7 - #15                 7 -       #20                                                                           - Asn Phe Thr Phe Ile Ser Gly Gln Leu Ser Ph - #e Ala Ser Ile Asp Gly         #               735                                                           - Gln Ser Asn Phe Pro Ser Ile Asn Glu Leu Se - #r Glu His Gly Trp Trp         #           750                                                               - Gly Ser Ala Asn Val Thr Ile Gln Glu Gly As - #n Asp Val Phe Lys Glu         #       765                                                                   - Asn Tyr Val Thr Leu Pro Gly Thr Phe Asn Gl - #u Cys Tyr Pro Asn Tyr         #   780                                                                       - Leu Tyr Gln Lys Ile Gly Glu Ser Glu Leu Ly - #s Ala Tyr Thr Arg Tyr         785                 7 - #90                 7 - #95                 8 -       #00                                                                           - Gln Leu Arg Gly Tyr Ile Glu Asp Ser Gln As - #p Leu Glu Ile Tyr Leu         #               815                                                           - Ile Arg Tyr Asn Ala Lys His Glu Thr Leu As - #p Val Pro Gly Thr Asp         #           830                                                               - Ser Leu Trp Pro Leu Ser Val Glu Ser Pro Il - #e Gly Arg Cys Gly Glu         #       845                                                                   - Pro Asn Arg Cys Ala Pro His Phe Glu Trp As - #n Pro Asp Leu Asp Cys         #   860                                                                       - Ser Cys Arg Asp Gly Glu Arg Cys Ala His Hi - #s Ser His His Phe Thr         865                 8 - #70                 8 - #75                 8 -       #80                                                                           - Leu Asp Ile Asp Val Gly Cys Thr Asp Leu Hi - #s Glu Asn Leu Gly Val         #               895                                                           - Trp Val Val Phe Lys Ile Lys Thr Gln Glu Gl - #y Tyr Ala Arg Leu Gly         #           910                                                               - Asn Leu Glu Phe Ile Glu Glu Lys Pro Leu Il - #e Gly Glu Ala Leu Ser         #       925                                                                   - Arg Val Lys Arg Ala Glu Lys Lys Trp Arg As - #p Lys Arg Glu Lys Leu         #   940                                                                       - Gln Leu Glu Thr Lys Arg Val Tyr Thr Glu Al - #a Lys Glu Ala Val Asp         945                 9 - #50                 9 - #55                 9 -       #60                                                                           - Ala Leu Phe Val Asp Ser Gln Tyr Asp Gln Le - #u Gln Ala Asp Thr Asn         #               975                                                           - Ile Gly Met Ile His Ala Ala Asp Lys Leu Va - #l His Arg Ile Arg Glu         #           990                                                               - Ala Tyr Leu Ser Glu Leu Pro Val Ile Pro Gl - #y Val Asn Ala Glu Ile         #      10050                                                                  - Phe Glu Glu Leu Glu Gly His Ile Ile Thr Al - #a Met Ser Leu Tyr Asp         #  10205                                                                      - Ala Arg Asn Val Val Lys Asn Gly Asp Phe As - #n Asn Gly Leu Thr Cys         #               10401030 - #                1035                              - Trp Asn Val Lys Gly His Val Asp Val Gln Gl - #n Ser His His Arg Ser         #              10550                                                          - Asp Leu Val Ile Pro Glu Trp Glu Ala Glu Va - #l Ser Gln Ala Val Arg         #          10705                                                              - Val Cys Pro Gly Arg Gly Tyr Ile Leu Arg Va - #l Thr Ala Tyr Lys Glu         #      10850                                                                  - Gly Tyr Gly Glu Gly Cys Val Thr Ile His Gl - #u Ile Glu Asn Asn Thr         #  11005                                                                      - Asp Glu Leu Lys Phe Lys Asn Cys Glu Glu Gl - #u Glu Val Tyr Pro Thr         #               11201110 - #                1115                              - Asp Thr Gly Thr Cys Asn Asp Tyr Thr Ala Hi - #s Gln Gly Thr Ala Ala         #              11350                                                          - Cys Asn Ser Arg Asn Ala Gly Tyr Glu Asp Al - #a Tyr Glu Val Asp Thr         #          11505                                                              - Thr Ala Ser Val Asn Tyr Lys Pro Thr Tyr Gl - #u Glu Glu Thr Tyr Thr         #      11650                                                                  - Asp Val Arg Arg Asp Asn His Cys Glu Tyr As - #p Arg Gly Tyr Val Asn         #  11805                                                                      - Tyr Pro Pro Val Pro Ala Gly Tyr Val Thr Ly - #s Glu Leu Glu Tyr Phe         #               12001190 - #                1195                              - Pro Glu Thr Asp Thr Val Trp Ile Glu Ile Gl - #y Glu Thr Glu Gly Lys         #              12150                                                          - Phe Ile Val Asp Ser Val Glu Leu Leu Leu Me - #t Glu Glu                     #           1225                                                              - (2) INFORMATION FOR SEQ ID NO:5:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 20 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: circular                                              -     (ii) MOLECULE TYPE: DNA (genomic)                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                 # 20               GATG                                                       __________________________________________________________________________

We claim:
 1. A transformed plant comprising a promoter that functions inplants operatively associated with a gene having a nucleotide basesequence coding for SEQ ID NO:2, or an insecticidal portion thereof. 2.A transformed plant according to claim 1 wherein the gene has thenucleotide base sequence of bases 99-3602 of SEQ ID NO:1 or a portionthereof encoding an insecticidal portion of SEQ ID NO:2.
 3. Atransformed plant comprising a promoter that functions in plantsoperatively associated with a gene having a nucleotide base sequencecoding for SEQ ID NO:4, or an insecticidal portion thereof.
 4. Atransformed plant according to claim 3 wherein the gene has thenucleotide base sequence of bases 67-3756 of SEQ ID NO:3 or a portionthereof encoding an insecticidal portion of SEQ ID NO:4.